Antibody

ABSTRACT

It has been demonstrated that certain compounds bind to TNF and stabilise a conformation of trimeric TNF that binds to the TNF receptor. Antibodies which selectively bind to complexes of such compounds with TNF superfamily members are disclosed. These antibodies may be used to detect further compounds with the same activity, and as target engagement biomarker.

FIELD OF THE INVENTION

This invention relates to antibodies which may be used to screen forsmall molecule modulators of the TNF superfamily that form complexeswith TNF superfamily members. In particular, the invention relates toantibodies which selectively bind to such complexes, and uses of suchantibodies. The present invention also relates to assays for identifyingnew modulators of the TNF superfamily using said antibodies.

BACKGROUND OF THE INVENTION

The Tumour Necrosis Factor (TNF) superfamily is a family of proteinsthat share a primary function of regulating cell survival and celldeath. Members of the TNF superfamily share a common core motif, whichconsists of two antiparallel β-pleated sheets with antiparallelβ-strands, forming a “jelly roll” β-structure. Another common featureshared by members of the TNF superfamily is the formation of homo- orheterotrimeric complexes. It is these trimeric forms of the TNFsuperfamily members that bind to, and activate, specific TNF superfamilyreceptors.

TNFα is the archetypal member of the TNF superfamily. Dysregulation ofTNFα production has been implicated in a number of pathologicalconditions of significant medical importance. For example, TNFα has beenimplicated in rheumatoid arthritis, inflammatory bowel diseases(including Crohn's disease), psoriasis, Alzheimer's disease (AD),Parkinson's disease (PD), pain, epilepsy, osteoporosis, asthma, systemiclupus erythematosus (SLE) and multiple sclerosis (MS). Other members ofthe TNF superfamily have also been implicated in pathologicalconditions, including autoimmune disease.

Conventional antagonists of TNF superfamily members are macromolecularand act by inhibiting the binding of the TNF superfamily member to itsreceptor. Examples of conventional antagonists include anti-TNFαantibodies, particularly monoclonal antibodies, such as infliximab(Remicade®), adalimumab (Humira®) and certolizumab pegol (Cimzia®), orsoluble TNFα receptor fusion proteins, such as etanercept (Enbrel®).

SUMMARY OF THE INVENTION

The present inventors have identified classes of small molecularentities (SME) that modulate TNFα. These compounds act by binding to thehomotrimeric form of TNFα, and inducing and/or stabilising aconformational change in the homotrimer of TNFα. For example,homotrimers of TNFα with the compound bound can bind to TNFα receptors,but are less able, or unable, to initiate signalling downstream of theTNFα receptor. These compounds can be used in the treatment ofconditions mediated by TNFα.

The present inventors have developed antibodies that bind selectively tocomplexes comprising such compounds and a TNF superfamily member. Theseantibodies may be used to identify further compounds that are capable ofinhibiting TNFα in this manner, and may also be used as targetengagement biomarkers.

Accordingly, the present invention provides an antibody that selectivelybinds to a complex comprising (i) a trimeric protein that is a TNFsuperfamily member and (ii) a compound that is capable of binding to atrimeric protein that is a TNF superfamily member, whereby thecompound-trimer complex binds to the requisite TNF superfamily receptorand modulates the signalling induced by the trimer through the receptor.

The present invention also provides an antibody that selectively bindsto a complex comprising (i) a human TNFα and (ii) a compound selectedfrom the group consisting of compounds (1)-(6), or salts or solvatesthereof.

The invention further provides

-   -   An antibody which competes for binding to TNFα with, or binds to        the same epitope on TNFα as, other antibodies of the invention.    -   An isolated polynucleotide encoding an antibody of the        invention.    -   An antibody of the invention for use in a method of treatment of        the human or animal body by therapy.    -   A pharmaceutical composition comprising an antibody of the        invention and a pharmaceutically acceptable adjuvant and/or        carrier.    -   Use of an antibody of the invention as a target engagement        biomarker for the detection of a compound-trimer complex in a        sample obtained from a subject; wherein said antibody is        detectable and said complex comprises a trimeric protein that is        a TNF superfamily member and a compound that is capable of        binding to a trimeric protein that is a TNF superfamily member,        whereby the compound-trimer complex binds to the requisite TNF        superfamily receptor and modulates the signalling induced by the        trimer through the receptor.    -   A method of detecting target engagement of a compound to a        trimeric TNF superfamily member, whereby the compound-trimer        complex binds to the requisite receptor and modulates the        signalling induced by the trimer through the receptor, said        method comprising:        -   (a) obtaining a sample from a subject administered said            compound;        -   (b) contacting an antibody of the invention to said sample            and a control sample, wherein said antibody is detectable;        -   (c) determining the amount of binding of said detectable            antibody to said sample and said control sample,    -   wherein binding of said detectable antibody to said sample        greater than binding of said detectable antibody to said control        sample indicates target engagement of said compound to said        trimeric TNF superfamily member.    -   Use of an antibody of the invention in screening for a compound        that elicits a conformational change in a trimeric TNF        superfamily member, wherein said conformational change modulates        the signalling of the requisite TNF superfamily receptor on        binding of the trimeric TNF superfamily member.    -   A complex comprising a trimeric protein that is a TNF        superfamily member and a compound that is bound thereto, whereby        the compound-trimer complex binds to the requisite TNF        superfamily receptor and modulates the signalling induced by the        trimer through the receptor, wherein said complex binds to an        antibody of the invention with a K_(D-ab) of 1 nM or less.    -   A TNFα trimer, said TNFα trimer being able to bind TNFR1, but        wherein signalling from said bound TNFR1 is attenuated or        antagonised, wherein said TNFα trimer binds to either or both of        the following antibodies with a a K_(D-ab) of 1 nM or less:        -   (i) an antibody with a heavy chain of SEQ ID NO: 27 and a            light chain of SEQ ID NO: 26; or        -   (ii) an antibody with a heavy chain of SEQ ID NO: 12 and a            light chain of SEQ ID NO: 11.    -   A compound that is capable of binding to a trimeric protein that        is a TNF superfamily member to form a complex, whereby the        compound-trimer complex binds to the requisite TNF superfamily        receptor and modulates the signalling induced by the trimer        through the receptor, wherein the compound-trimer complex binds        to an antibody of the invention with a K_(D-ab) of 1 nM or less.    -   A complex as defined above, a trimer as defined above, or a        compound according as defined above for use in a method of        therapy practised on the human or animal body.    -   A method of identifying a compound that is capable of binding to        a trimeric protein that is a TNF superfamily member and        modulating signalling of the trimeric protein through the        receptor, comprising the steps of:        -   (a) performing a binding assay to measure the binding            affinity of a test compound-trimer complex comprising a            trimeric protein that is a TNF superfamily member and a test            compound to an antibody that selectively binds to said            complex;        -   (b) comparing the binding affinity as measured in step (a)            with the binding affinity of a different compound-trimer            complex known to bind with high affinity to the antibody            referred to in step (a); and        -   (c) selecting the compound present in the compound-trimer            complex of step (a) if its measured binding affinity is            acceptable when considered in the light of the comparison            referred to in step (b).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 highlights residues N168, 1194, F220 and A221 on the crystalstructure of human TNFα.

FIG. 2 shows results of HPLC experiments with the CA185_01974 mFab andcompound (1). Peaks corresponding to excess Fab appear at a 1.5× and2.0× excess. The stoichiometry was therefore determined to be 1 Fab: 1TNFα trimer.

FIG. 3 shows results of HPLC experiments with the CA185_01979 mFab andcompound (1). Again, peaks corresponding to excess Fab appear at a 1.5×and 2.0× excess. The stoichiometry was therefore also determined to be 1Fab: 1 TNFα trimer.

FIG. 4 presents results of total TNFα ELISA with compounds (3), (4) and(5) using a commercial anti-TNFα polyclonal antibody.

FIG. 5 presents results of conformation specific TNFα ELISA withCA185_01974.0 and compounds (3), (4) and (5). Apo TNFα gave no signal inthis assay, demonstrating the specific nature of the binding of antibodyCA185_01974 to compound-bound TNFα.

FIG. 6 shows FACS histogram plots of staining with CA185_01974 andCA185_01979 at 1 and 10 μg/ml. These plots demonstrate that theantibodies only recognise TNFα which has been pre-incubated withcompound (1). There is no staining with the DMSO control.

FIG. 7 shows FACS histogram plots of staining with CA185_01974 for aparental NS0 cell line and an engineered NS0 cell line, whichoverexpresses membrane TNFα. Cells were incubated with compound (1) orDMSO and stained with the antibody Fab fragment. Again, results indicateno staining for the DMSO control (for either the parental or engineeredcell line). In the presence of compound (1) staining is, however,observed for the engineered cell line.

FIG. 8 shows sensograms for the determination of affinity values forCA185_01974 using cynomolgus TNFα. Controls (top panels) containedcynomolgus TNFα and DMSO. The bottom panels then present duplicatedexperiments for cynomolgus TNFα complexed with compound (4).

FIG. 9 shows sensograms for the determination of affinity values forCA185_01974 using human TNFα. Controls (top panels) contained human TNFαand DMSO. The bottom panels then present duplicated experiments forhuman TNFα complexed with compound (4).

FIG. 10 shows the structures of compounds (1)-(6).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the LCDR1 of CA185_01974.0.

SEQ ID NO: 2 shows the LCDR2 of CA185_01974.0.

SEQ ID NO: 3 shows the LCDR3 of CA185_01974.0.

SEQ ID NO: 4 shows the HCDR1 of CA185_01974.0.

SEQ ID NO: 5 shows the HCDR2 of CA185_01974.0.

SEQ ID NO: 6 shows the HCDR3 of CA185_01974.0.

SEQ ID NO: 7 shows the amino acid sequence of the LCVR of CA185_01974.0.

SEQ ID NO: 8 shows the amino acid sequence of the HCVR of CA185_01974.0.

SEQ ID NO: 9 shows the DNA sequence of the LCVR of CA185_01974.0.

SEQ ID NO: 10 shows the DNA sequence of the HCVR of CA185_01974.0.

SEQ ID NO: 11 shows the amino acid sequence of the kappa light chain ofCA185_01974.0.

SEQ ID NO: 12 shows the amino acid sequence of the mIgG1 heavy chain ofCA185_01974.0.

SEQ ID NO: 13 shows the amino acid sequence of the mFab (no hinge) heavychain of CA185_01974.0.

SEQ ID NO: 14 shows the DNA sequence of the kappa light chain ofCA185_01974.0.

SEQ ID NO: 15 shows the DNA sequence of the mIgG1 heavy chain ofCA185_01974.0.

SEQ ID NO: 16 shows the DNA sequence of the mFab (no hinge) heavy chainof CA185_01974.0.

SEQ ID NO: 17 shows the LCDR2 of CA185_01979.0.

SEQ ID NO: 18 shows the LCDR3 of CA185_01979.0.

SEQ ID NO: 19 shows the HCDR1 of CA185_01979.0.

SEQ ID NO: 20 shows the HCDR2 of CA185_01979.0.

SEQ ID NO: 21 shows the HCDR3 of CA185_01979.0.

SEQ ID NO: 22 shows the amino acid sequence of the LCVR ofCA185_01979.0.

SEQ ID NO: 23 shows the amino acid sequence of the HCVR ofCA185_01979.0.

SEQ ID NO: 24 shows the DNA sequence of the LCVR of CA185_01979.0.

SEQ ID NO: 25 shows the DNA sequence of the HCVR of CA185_01979.0.

SEQ ID NO: 26 shows the amino acid sequence of the kappa light chain ofCA185_01979.0.

SEQ ID NO: 27 shows the amino acid sequence of the mIgG1 heavy chain ofCA185_01979.0.

SEQ ID NO: 28 shows the amino acid sequence of the mFab (no hinge) heavychain of CA185_01979.0.

SEQ ID NO: 29 shows the DNA sequence of the kappa light chain ofCA185_01979.0.

SEQ ID NO: 30 shows the DNA sequence of the mIgG1 heavy chain ofCA185_01979.0.

SEQ ID NO: 31 shows the DNA sequence of the mFab (no hinge) heavy chainof CA185_01979.0.

SEQ ID NO: 32 shows the amino acid sequence of rat TNFα.

SEQ ID NO: 33 shows the amino acid sequence of mouse TNFα.

SEQ ID NO: 34 shows the amino acid sequence of human TNFα.

SEQ ID NO: 35 shows the amino acid sequence of the soluble form of humanTNFα.

SEQ ID NO: 36 shows the amino acid sequence of the soluble form of humanTNFα, but without the initial “S” (which is a cloning artefact in SEQ IDNO: 35)

DETAILED DESCRIPTION OF THE INVENTION Modulators of TNF SuperfamilyMembers

The present inventors have identified test compounds that bind totrimeric forms of the TNF superfamily members. These compounds are smallmolecular entities (SMEs) that have a molecular weight of 1000 Da orless, generally 750 Da or less, more suitably 600 Da or less. Themolecular weight may be in the range of about 50-about 1000 Da, or about100-about 1000 Da. These compounds stabilise a conformation of thetrimeric TNF superfamily member that binds to the requisite TNFsuperfamily receptor and modulate the signalling of the receptor.Examples of such compounds include compounds of formulae (1)-(6).

The stabilising effect of compounds of the invention on trimeric formsof TNF superfamily members may be quantified by measuring the thermaltransition midpoint (Tm) of the trimers in the presence and absence ofthe compound. Tm signifies the temperature at which 50% of thebiomolecules are unfolded. Compounds which stabilise TNF superfamilymember trimers will increase the Tm of the trimers. Tm may be determinedusing any appropriate technique known in the art, for example usingdifferential scanning calorimetry (DSC) or fluorescence probed thermaldenaturation assays.

The compounds may bind inside the central space present within the TNFsuperfamily member trimer (i.e. the core of the trimer).

These compounds may turn the TNF superfamily member into a TNFsuperfamily receptor antagonist. These compounds are therefore capableof blocking the TNF superfamily member signalling without having tocompete with the high affinity interaction between the TNF superfamilymember and its receptor.

Alternatively, the compounds may stabilise a conformation of thetrimeric TNF superfamily member that binds to the requisite TNFsuperfamily receptor and enhances the signalling of the receptor. Thesecompounds are therefore capable of increasing the TNF superfamily membersignalling without having to compete with the high affinity interactionbetween the TNF superfamily member and its receptor.

Where herein the compounds are described as antagonists, it will beunderstood that the compounds may equally be agonists and increasesignalling by a TNF superfamily receptor that is bound to a complex of aTNF superfamily member trimer and such an agonist compound. Similarly,where other disclosure refers to antagonistic compounds, methods ofidentifying such compounds and uses of such compounds, this disclosuremay refer equally to agonist compounds.

The compounds described herein are allosteric modulators that bind tothe natural agonists of the TNF superfamily receptors, i.e. to trimericforms of TNF superfamily members and drive these trimers to adopt aconformation that still binds to the requisite TNF superfamily receptorand modulates signalling by the receptor. By modulating, it will beunderstood that the compound may have an antagonistic effect and sodecrease signalling by a TNF superfamily receptor, or else a stimulatoryeffect and so increase or enhance signalling by a TNF superfamilyreceptor.

These compounds may convert the natural TNF superfamily member agonistsinto antagonists. In contrast, conventional TNF superfamily memberantagonists bind to the TNF superfamily member or the TNF superfamilyreceptor and prevent the binding of the TNF superfamily member to therequisite receptor. In the alternative, the compounds may increasesignalling by a TNF superfamily receptor when the TNF superfamily memberis bound compared to the level of signalling by the TNF superfamilyreceptor when the TNF superfamily member is bound in the absence of thecompound. The compounds may therefore convert the natural TNFsuperfamily member agonists into so-called “super-agonists”. Thecompounds may therefore also be known as allosteric modulators of ligandactivity (AMLAs).

The compounds are not limited in terms of their chemical formula orstructure, provided that they bind to at least one TNF superfamilymember and stabilise a conformation of the trimeric TNF superfamilymember that binds to the requisite TNF superfamily receptor and modulatethe signalling of the TNF superfamily receptor. The compounds cantherefore be identified using the antibodies and methods describedherein. The compounds may comprise a benzimidazole moiety or an isosterethereof.

The compounds may increase the binding affinity of TNF superfamilymembers (in the form of a compound-trimer complex) to the requisitereceptor compared to the binding affinity of the TNF superfamily membersto the requisite receptor in the absence of the compounds.

The compounds bind to the trimeric forms of TNF superfamily members.Such compounds may bind specifically (or selectively) to the trimericforms of one or more TNF superfamily members. A compound may bindspecifically (or selectively) to only one of the TNF superfamilymembers, but not to any other TNF superfamily members. A compound mayalso bind specifically to two, three, four or up to all of the TNFsuperfamily members. By specific (or selective), it will be understoodthat the compounds bind to the molecule or molecules of interest, inthis case the trimeric form of the TNF superfamily member, with nosignificant cross-reactivity to any other molecule, which may includeother members of the TNF superfamily. Cross-reactivity may be assessedby any suitable method, for example surface plasmon resonance.Cross-reactivity of a compound for the trimeric form of a TNFsuperfamily member with a molecule other than the trimeric form of thatparticular TNF superfamily member may be considered significant if thecompound binds to the other molecule at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or100% as strongly as it binds to the trimeric form of the TNF superfamilymember of interest. For example, cross reactivity may be consideredsignificant if the compound binds to the other molecule about 5%-about100%, typically about 20%-about 100%, or about 50%-about 100% asstrongly as it binds to the trimeric form of the TNF superfamily memberof interest. A compound that is specific (or selective) for the trimericform of a TNF superfamily member may bind to another molecule at lessthan about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25% or 20% the strength that it binds to the trimeric form of theTNF superfamily member (down to zero binding). The compound suitablybinds to the other molecule at less than about 20%, less than about 15%,less than about 10% or less than about 5%, less than about 2% or lessthan about 1% the strength that it binds to the trimeric form of the TNFsuperfamily member (down to zero binding).

The rates at which a test compound binds to a TNF superfamily member isreferred to herein as the “on” rate” k_(on-c) and the rate at which thetest compound dissociates from the TNF superfamily member is referred toherein as the “off” rate or k_(off-c). As used herein, the symbol“K_(D-c)” denotes the binding affinity (dissociation constant) of a testcompound for a TNF superfamily member. K_(D-c) is defined ask_(off-c)/k_(on-c). Test compounds may have slow “on” rates, which canbe measured in minutes by mass spectral analysis of the TNF superfamilymember and compound-trimer complex peak intensities. K_(D-c) values fora test compound can be estimated by repeating this measurement atdifferent TNF superfamily member: compound-trimer complex ratios.Typically, binding of compounds of the invention to TNF superfamilytrimers is characterized by fast “on” rates, ideally about 10⁷M⁻¹s⁻¹,with slow “off” rate, for example values typically of 10⁻³ s⁻¹, 10⁻⁴s⁻¹, or no measurable “off” rate.

As used herein, the symbol “k_(on-r)” denotes the rate (the “on” rate)at which a compound-trimer complex binds to a TNF superfamily receptor.As used herein, the symbol “k_(off-r)” denotes the rate (the “off” rate)at which a compound-trimer complex dissociates from a TNF superfamilyreceptor. As used herein, the symbol “K_(D-r)” denotes the bindingaffinity (dissociation constant) of a compound-trimer complex for asuperfamily receptor. K_(D-r) is defined as k_(off-r)/k_(on-r).

The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the test compound (i.e. in the form of acompound-trimer complex) may be at least about 1.5 times, 2 times, 3times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50times, 60 times, 70 times, 80 times, 90 times, 100 times lower than theK_(D-r) value of the TNF superfamily member for binding to its receptorin the absence of the test compound. The K_(D-r) value of thecompound-trimer complex for binding to the TNF superfamily member may bedecreased at least about 1.5 times, generally at least about 3 times,more suitably at least about 4 times the K_(D-r) value of the TNFsuperfamily trimer binding to the TNF superfamily receptor in theabsence of the test compound, i.e. the binding affinity of thecompound-trimer complex for the TNF superfamily may be increased atleast about 1.5-fold, generally at least about three-fold, more suitablyat least about four-fold compared to the binding affinity of the TNFsuperfamily trimer to the TNF superfamily receptor in the absence oftest compound.

A compound described herein may increase the binding affinity of the TNFsuperfamily member to its receptor by about 2 times, 3 times, 4 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times or more compared to the bindingaffinity of the TNF superfamily member to its receptor in the absence ofthe compound.

The binding affinity may be given in terms of binding affinities(K_(D-r)) and may be given in any appropriate units, such as μM, nM orpM. The smaller the K_(D-r) value, the larger the binding affinity ofthe TNF superfamily member to its receptor.

The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the compound may be at least about 1.5times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times,40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 timeslower than the K_(D-r) value of the TNF superfamily member for bindingto its receptor in the absence of the test compound.

The decrease in the K_(D-r) value of the compound-trimer complex forbinding to the TNF superfamily receptor compared to the K_(D-r) value ofthe TNF superfamily trimer alone binding to the TNF superfamily receptormay result from an increase in the on rate (k_(on-r)) of thecompound-trimer complex binding to the TNF superfamily receptor comparedto the TNF superfamily trimer alone, and/or a decrease in the off rate(k_(off-r)) compared to the TNF superfamily trimer alone. The on rate(k_(on-r)) of the compound-trimer complex binding to the TNF superfamilyreceptor is generally increased compared to the TNF superfamily trimeralone. The off rate (k_(off-r)) of the compound-trimer complex bindingto the TNF superfamily receptor is generally decreased compared to theTNF superfamily trimer alone. Most suitably, the on rate (k_(on-r)) ofthe compound-trimer complex binding to the TNF superfamily receptor isincreased, and the off-rate (k_(off-r)) of the compound-trimer complexbinding to the TNF superfamily receptor is decreased, compared to theTNF superfamily trimer alone. The k_(on-r) value of the compound-trimercomplex to the requisite TNF superfamily receptor may be increased by atleast about 1.5-fold or at least about two-fold and suitably at leastabout three fold compared to the k_(on-r) value of the TNF superfamilytrimer binding to its receptor in the absence of the compound and/or thek_(off-r) value of the compound-trimer complex to the requisite TNFsuperfamily receptor may be decreased by at least about 1.2-fold, atleast about 1.6-fold, at least about two-fold, more suitably at leastabout 2.4-fold compared to the k_(off-r) value of the TNF superfamilytrimer binding to its receptor in the absence of the compound.

The on-rate for compound binding to TNF superfamily trimer (k_(on-c)) istypically faster than the on-rate for compound-trimer complex binding toTNF superfamily receptor (k_(on-r)). The off-rate for compound-trimercomplex binding to TNF superfamily receptor (k_(off-r)) is alsotypically faster than the off-rate for compound binding to TNFsuperfamily trimer (k_(off-c)). Most suitably, the on-rate for compoundbinding to TNF superfamily trimer (k_(on-c)) is faster than the on-ratefor compound-trimer complex binding to TNF superfamily receptor(k_(on-r)), and the off-rate for compound-trimer complex binding to TNFsuperfamily receptor (k_(off-r)) is faster than the off-rate forcompound binding to TNF superfamily trimer (k_(off-c)). The K_(D-c)value of the compound for binding to TNF superfamily trimer is generallylower than the K_(D-r) value of the compound-trimer complex for bindingto TNF superfamily receptor, i.e. the compound has a higher affinity forthe trimer than the compound-trimer complex has for the receptor.

The k_(on-r), k_(off-r), and K_(D-r) values for both the compound-trimercomplex and the TNF superfamily trimer to the requisite TNF superfamilyreceptor may be determined using any appropriate technique, for examplesurface plasmon resonance, mass spectrometry and isothermal calorimetry.The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the test compound may be 1 μM, 100 nM, 10nM, 5 nM, 1 nM, 100 pM, 10 pM or less (typically down to a lower valueof about 1 pM). The K_(D-r) value of the TNF superfamily member forbinding to its receptor in the presence of the test compound (i.e. in acompound-trimer complex) may be 1 nM or less. The K_(D-r) value of acompound-trimer complex for binding to the requisite TNF superfamilyreceptor may be less than 600 pM, more preferably less than 500 pM, lessthan 400 pM, less than 300 pM, less than 200 pM, less than 100 pM orless than 50 pM (again down to a lower value of about 1 pM). The K_(D-r)value of a compound-trimer complex for binding to the requisite TNFsuperfamily receptor may be less than about 200 pM (to about 1 pM).

Compounds may be identified by an assay which comprises determining theK_(D-r) of the trimeric form of the TNF superfamily member in a sampleof the TNF superfamily member and the compound; comparing the K_(D-r) ofthe trimeric form of the TNF superfamily member in the sample with acontrol sample; and selecting a compound.

The compounds stabilise the trimeric form of the TNF superfamily member.Stabilisation is considered to occur if a test compound increases theproportion of trimer compared to the amount of trimer observed for asample containing the TNF superfamily member and the destabilising agentin the absence of the test compound. The test compound may increase theamount of trimer by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%,400% or more compared to the amount of trimer present in a samplecontaining the TNF superfamily member and the destabilising agent in theabsence of the test compound.

The test compound may also increase the amount of trimer compared tothat observed for a sample of the TNF superfamily member in the absenceof both the destabilising agent and the test compound. The test compoundmay increase the amount of trimer by about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%,200%, 300%, 400% or more compared to the amount of trimer present in asample containing the TNF superfamily member in the absence of both thedestabilising agent and the test compound.

The test compound may increase the amount of the TNF superfamily memberbound to its receptor by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,300%, 400% or more compared to the amount of the TNF superfamily memberbound to its receptor in a sample containing the TNF superfamily memberin the absence of the test compound.

The test compounds may enhance the stability of the trimeric form of theTNF superfamily member. Enhanced stability of the trimeric form of theTNF superfamily member is considered to occur if a test compoundincreases the thermal transition midpoint (T_(m)) of the trimeric formof the TNF superfamily member compared to the T_(m) of the trimeric formof the TNF superfamily member observed for a sample containing the TNFsuperfamily member and the destabilising agent in the absence of thetest compound. The T_(m) of the trimeric form of the TNF superfamilymember is the temperature at which 50% of the biomolecules are unfolded.The T_(m) of the trimeric form of the TNF superfamily member in thepresence and/or absence of the test compound may be measured using anyappropriate technique known in the art, for example using differentialscanning calorimetry (DSC) or fluorescence probed thermal denaturationassays.

The test compound may increase the T_(m) of the trimeric form of the TNFsuperfamily member by at least 1° C., at least 2° C., at least 5° C., atleast 10° C., at least 15° C., at least 20° C. or more compared to theT_(m) of the trimeric form of the TNF superfamily member in a samplecontaining the TNF superfamily member in the absence of the testcompound. The test compound may increase the T_(m) of the trimeric formof the TNF superfamily member by at least 1° C., typically by at least10° C. and more suitably by between 10° C. and 20° C.

The compounds may completely or partially inhibit signalling through aTNF receptor when a TNF superfamily member in the form of acompound-trimer complex binds to the receptor. The compound may act toreduce signalling through a TNF superfamily receptor by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Alternatively, thecompounds may increase signalling through a TNF receptor when a TNFsuperfamily member in the form of a compound-trimer complex binds to thereceptor. The compound may act to increase signalling through a TNFsuperfamily receptor by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100% or 200%. Any change in the level of signalling maybe measured by any appropriate technique, including measuring reportergene activity by alkaline phosphatase or luciferase, NF-κB translocationusing machines such as the Cellomics Arrayscan, phosphorylation ofdownstream effectors, recruitment of signalling molecules, or celldeath.

The compounds may modulate at least one of the downstream effects ofsignalling through a TNF receptor when a TNF superfamily member in theform of a compound-trimer complex binds to the receptor. Such effectsare discussed herein and include TNF superfamily-induced IL-8, IL17A/F,IL2 and VCAM production, TNF superfamily-induced NF-κB activation andneutrophil recruitment. Standard techniques are known in the art formeasuring the downstream effects of TNF superfamily members. Thecompounds may modulate at least 1, 2, 3, 4, 5, 10 or up to all of thedownstream effects of signalling through a TNF receptor.

The activity of the compounds may be quantified using standardterminology, such as IC₅₀ or half maximal effective concentration (EC₅₀)values. IC₅₀ values represent the concentration of a compound that isrequired for 50% inhibition of a specified biological or biochemicalfunction. EC₅₀ values represent the concentration of a compound that isrequired for 50% of its maximal effect. The compounds may have IC₅₀ orEC₅₀ values of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 100 pM or less(down to a lower value of about 10 pM or 1 pM). IC₅₀ and EC₅₀ values maybe measured using any appropriate technique, for example cytokineproduction can be quantified using ELISA. IC₅₀ and EC₅₀ values can thenbe generated using a standard 4-parameter logistic model also known asthe sigmoidal dose response model.

As mentioned above, examples of compounds which are capable of bindingto TNF and modulating signalling are compounds of formulae (1)-(6).

Modulator-TNF Superfamily Member Complexes

The present inventors have found that binding of the compounds describedherein to trimeric forms of TNF superfamily members results in aconformational change in the TNF superfamily trimer. In particular, theTNF superfamily member trimer takes on a deformed or distortedconformation when bound by a compound as disclosed herein.

For example, when compounds (1)-(6) are bound the soluble domain ofhuman TNFα the TNF retains its trimeric structure but the A and Csubunits move away from each other and C rotates to generate a cleftbetween these subunits.

Without being bound by theory, it is believed that, in the absence of acompound, trimeric TNF superfamily members, including trimeric TNFα, arecapable of binding to three separate dimeric TNF superfamily memberreceptors. Each of the dimeric TNF superfamily member receptors iscapable of binding to two separate TNF superfamily trimers. This resultsin the aggregation of multiple TNF superfamily member trimers and TNFsuperfamily member receptor dimers, creating signalling rafts thatinitiate downstream signalling.

When trimeric TNFα is bound to the compound, the conformation of theresulting complex is deformed. Accordingly, without being bound bytheory, it is believed that, in the presence of a compound as disclosedherein, trimeric TNF superfamily members, including trimeric TNFα, areonly capable of binding to two separate dimeric TNF superfamily memberreceptors. The fact that only two, rather than three, separate dimericTNF superfamily member receptors bind to the trimeric TNF superfamilymember reduces or inhibiting the aggregation of multiple TNF superfamilymember trimers and TNF superfamily member receptor dimers. This reducesor inhibits the formation of signalling rafts and so reduces or inhibitsdownstream signalling.

The antibodies of the invention may be used to detect TNF superfamilymembers with a distorted conformation as a result of the binding of acompound as disclosed herein. Typically the TNF superfamily member witha distorted or deformed conformation is a trimeric TNF superfamilymember. However, antibodies of the invention may also bind to otherforms of the TNF superfamily member. For example, antibodies of theinvention may bind to TNF superfamily monomers.

The TNF superfamily member is typically TNFα, and may be trimeric TNFα(particularly TNFα_(s)).

Accordingly, the invention provides a complex comprising a trimericprotein that is a TNF superfamily member and a compound that is boundthereto, whereby the compound-trimer complex binds to the requisite TNFsuperfamily receptor and modulates the signalling induced by the trimerthrough the receptor, wherein said complex binds to an antibody of theinvention with an affinity of at least 1 nM (i.e. 1 nM or less, down toabout 1 pM). The TNF superfamily member is typically TNFα, moreparticularly TNFα_(s).

Furthermore, the antibody generally binds to the complex with anaffinity of that is at least about 100 times lower (the affinity isimproved at least about 100 times), suitably about 200 times lower,relative to the affinity for binding to the compound in the absence ofthe TNF timer and/or for binding to the TNF trimer in the absence ofcompound.

The present invention further provides a compound that is capable ofbinding to a trimeric protein that is a TNF superfamily member to form acomplex, whereby the compound-trimer complex binds to the requisite TNFsuperfamily receptor and modulates the signalling induced by the trimerthrough the receptor, wherein the compound-trimer complex binds to anantibody of the invention with a K_(D-ab) of 1 nM or less (down to about1 pM). The TNF superfamily member is typically TNFα, most particularlyTNFα_(s).

The antibody typically binds to the complex with an affinity of that isat least about 100 times lower (the affinity is improved at least about100 times), more suitably about 200 times lower, relative to theaffinity for binding to the compound in the absence of the TNF timerand/or for binding to the TNF trimer in the absence of compound.

The compound-trimer complex may bind to any antibody of the invention.Particularly, the compound-trimer complex may bind to an antibodycomprising the amino acid sequences disclosed herein.

A compound or complex described herein may be used in the treatmentand/or prophylaxis of a pathological condition. Accordingly, provided isa compound or complex of the invention for use in a method of therapypracticed on the human or animal body. The invention also provides amethod of therapy comprising the administration of a compound or complexof the invention to a subject. The compound or complex of the inventionmay be used in any therapeutic indication and/or pharmaceuticalcomposition described herein.

Antibodies

The invention provides antibodies that selectively bind to at least onecompound-trimer complex comprising at least one compound disclosedherein and a trimeric TNF superfamily member.

Typically, selective binding of an antibody of the invention to acompound-(trimer) complex is measured relative to the binding of theantibody to the compound in the absence of the TNF superfamily member,or to the TNF superfamily member in the absence of the compound or toother (different) compound-(trimer) complexes.

In particular, the invention provides an antibody that selectively bindsto a complex comprising (i) a trimeric protein that is a TNF superfamilymember and (ii) a compound that is capable of binding to a trimericprotein that is a TNF superfamily member, whereby the compound-trimercomplex binds to the requisite TNF superfamily receptor and modulatesthe signalling induced by the trimer through the receptor. Typicallysaid antibody binds selectively to said complex relative to its bindingto the TNF superfamily member in the absence of the compound or to thecompound in the absence of the TNF superfamily member.

The compound may be any compound described above, including compounds(1)-(6) (or salts or solvates thereof). As discussed further below, theTNF superfamily member may be any of the superfamily members, but istypically TNFα. More particularly, the TNFα is human TNFα, especiallysoluble TNFα (TNFα_(s)). The TNFα_(s) may have the sequence of SEQ IDNO: 35 or SEQ ID NO: 36, or may be a variant of SEQ ID NO: 35 or SEQ IDNO: 36. Such variants typically retain at least about 60%, 70%, 80%,90%, 91%, 92%, 93%, 94% or 95% identity to SEQ ID NO: 35 or SEQ ID NO:36 (or even about 96%, 97%, 98% or 99% identity). In other words, suchvariants may retain about 60%-about 99% identity to SEQ ID NO:35 or SEQID NO:36, suitably about 80%-about 99% identity to SEQ ID NO:35 or SEQID NO:36, more suitably about 90%-about 99% identity to SEQ ID NO:35 orSEQ ID NO:36 and most suitably about 95%-about 99% identity to SEQ IDNO:35 or SEQ ID NO:36. Variants are described further below.

The term “corresponding sequence” indicates that the TNFα may have thewild-type amino sequence of any known animal or human TNFα, inparticular human TNFα, for instance SEQ ID NO: 36. It may be solubleTNFα (sTNFα) or membrane-bound TNFα, or both. Soluble homotrimeric TNFα(sTNF) is released from membrane-bound homotrimeric TNFα (mTNF) viaproteolytic cleavage by the metalloprotease TNF alpha converting enzyme(TACE/ADAM17; though other proteinases can also release sTNF such asADAM10, ADAM19, matrix metalloproteinase 7 and proteinase 3 which mayyield corresponding soluble TNFα sequences that may be extended ortruncated by 1, 2, 3, 4, or 5 amino acids relative to a TACE cleavedsTNFα such as SEQ ID NO: 36). The soluble 52 kDa trimeric sTNF takes ona triangular pyramid shape. A human sequence encompassed by the termmTNF is shown in SEQ ID NO: 34, and a human sequence encompassed by theterm sTNF (the product of the action of TACE on SEQ ID NO: 34) is shownin SEQ ID NO: 36. Corresponding sequences of rat and mouse mTNFα arepresented in SEQ ID NO:32 and 33, respectively. Corresponding sequencesof TNFα from other animals (or known variants of the human sequence) maybe readily overlaid with the SEQ ID NO:36 sequence and given the sameamino acid numbering as for SEQ ID NO:36 (used in the numbering of TNFαamino acids herein). For instance, the sequence from various animals maybe found within the Uniprot database (www.uniprot.org) including humansequences P01375 and Q5STB3. The corresponding sTNFα sequences may bethe 157 amino acid C-terminal end of the mTNFα sequence (as SEQ IDNO:36) or may be longer or shorter by one, two or three amino acids (therat and mouse sequences being 156 amino acids). The corresponding sTNFαsequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 amino acid substitutions relative to SEQ IDNO:36. The corresponding sTNFα sequence may have 80, 90, 95, 96, 97, 98,or 99% amino acid sequence identity to SEQ ID NO:36 over the length ofSEQ ID NO:36.

As discussed above, although the present disclosure generally relates tobinding of antibodies of the invention to TNF superfamily membertrimers, antibodies of the invention may also bind to other forms of theTNF superfamily member. To illustrate, the Examples of the presentapplication demonstrate that the CA185_0179 antibody binds to trimericTNF. However, as shown in FIG. 1 (crystal structure of the CA185_0179antibody bound to a TNFα monomer in the presence of compound (1)) theantibody also appears to bind to monomeric TNFα. Without being bound bytheory, in the presence of the compound it is believed that the solubledomain of the TNF retains its trimeric structure. However, the A and Csubunits move away from each other (and the C subunit rotates) togenerate a cleft between these two subunits. Thus although antibodies ofthe invention bind to distorted trimers, it is also possible that theantibodies can still bind if the trimeric structure is forced apart intomonomers.

With regards to the “A” and “C” subunits, when looking at a crystalstructure of a TNFα trimer from the side it is approximately shaped likea pyramid/cone. When you look down the trimer axis with the N- andC-termini of the monomer ends pointing towards you then you are lookingat the “fat” end of the trimer. In the distorted structure withcompound, a cleft opens between A and C subunits in which, without beingbound by theory, the Ab of the invention binds.

Which chain is A, B or C may be ascertained by measuring three distancesbetween three C-alpha atoms of three identical residues—e.g. P117 ineach chain (G121 is also appropriate).

The three distances form a triangle which is equilateral in apo TNF butdistorted when compound is bound. The shortest distance is between BCand the longest between AC (for instance AC=13.8 Å, AB=12.3 Å, BC=10.2Å); thus looking down through the axis of the molecule with N/C terminipointing towards you the longest distance defines C then A chains goinganti-clockwise, then B and C again continuing anti-clockwise.

The invention therefore also provides antibodies that selectively bindto a complex comprising human TNFα and a compound selected from thegroup consisting of compounds (1)-(6), or salts or solvates thereof. Thehuman TNFα is typically soluble TNFα (TNFα_(s)). The TNFα_(s) maycomprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36, or a variantthereof. Such variants may retain at least about 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:35or SEQ ID NO: 36 (see above and methods of identifying variants aredescribed below). The TNFα may be trimeric.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An antibody refers to a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asHCVR or V_(H)) and a heavy chain constant region. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor V_(L)) and a light chain constant region. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR).

The constant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

An antibody of the invention may be a monoclonal antibody or apolyclonal antibody, and will typically be a monoclonal antibody. Anantibody of the invention may be a chimeric antibody, a CDR-graftedantibody, a nanobody, a human or humanised antibody or anantigen-binding portion of any thereof. For the production of bothmonoclonal and polyclonal antibodies, the experimental animal istypically a non-human mammal such as a goat, rabbit, rat or mouse butthe antibody may also be raised in other species.

Polyclonal antibodies may be produced by routine methods such asimmunisation of a suitable animal, with the antigen of interest. Bloodmay be subsequently removed from the animal and the IgG fractionpurified.

Antibodies generated against compound-trimer complexes of the inventionmay be obtained, where immunisation of an animal is necessary, byadministering the polypeptides to an animal, e.g. a non-human animal,using well-known and routine protocols, see for example Handbook ofExperimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell ScientificPublishers, Oxford, England, 1986). Many warm-blooded animals, such asrabbits, mice, rats, sheep, cows, camels or pigs may be immunized.However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies of the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by for example the methods describedby Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551; WO2004/051268 and WO2004/106377.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

Fully human antibodies are those antibodies in which the variableregions and the constant regions (where present) of both the heavy andthe light chains are all of human origin, or substantially identical tosequences of human origin, but not necessarily from the same antibody.Examples of fully human antibodies may include antibodies produced, forexample by the phage display methods described above and antibodiesproduced by mice in which the murine immunoglobulin variable andoptionally the constant region genes have been replaced by their humancounterparts e.g. as described in general terms in EP 0546073, U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,EP 0438474 and EP 0463151.

Alternatively, an antibody according to the invention may be produced bya method comprising: immunising a non-human mammal with an immunogencomprising a compound-trimer complex of a trimeric TNF superfamilymember and a compound disclosed herein; obtaining an antibodypreparation from said mammal; deriving therefrom monoclonal antibodiesthat selectively recognise said complex and screening the population ofmonoclonal antibodies for monoclonal antibodies that bind to the TNFsuperfamily member only in the presence of the compound.

The antibody molecules of the present invention may comprise a completeantibody molecule having full length heavy and light chains or afragment or antigen-binding portion thereof. The term “antigen-bindingportion” of an antibody refers to one or more fragments of an antibodythat retain the ability to selectively bind to an antigen. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. The antibodies and fragments andantigen binding portions thereof may be, but are not limited to Fab,modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, single domain antibodies(e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies,Bis-scFv, diabodies, triabodies, tetrabodies and epitope-bindingfragments of any of the above (see for example Holliger and Hudson,2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, DrugDesign Reviews—Online 2(3), 209-217). The methods for creating andmanufacturing these antibody fragments are well known in the art (seefor example Verma et al., 1998, Journal of Immunological Methods, 216,165-181). Other antibody fragments for use in the present inventioninclude the Fab and Fab′ fragments described in International patentapplications WO 2005/003169, WO 2005/003170 and WO 2005/003171 andFab-dAb fragments described in International patent applicationWO2009/040562. Multi-valent antibodies may comprise multiplespecificities or may be monospecific (see for example WO 92/22853 and WO05/113605). These antibody fragments may be obtained using conventionaltechniques known to those of skill in the art, and the fragments may bescreened for utility in the same manner as intact antibodies.

The constant region domains of the antibody molecule of the presentinvention, if present, may be selected having regard to the proposedfunction of the antibody molecule, and in particular the effectorfunctions which may be required. For example, the constant regiondomains may be human IgA, IgD, IgE, IgG or IgM domains. In particular,human IgG constant region domains may be used, especially of the IgG1and IgG3 isotypes when the antibody molecule is intended for therapeuticuses and antibody effector functions are required. Alternatively, IgG2and IgG4 isotypes may be used when the antibody molecule is intended fortherapeutic purposes and antibody effector functions are not required.

An antibody of the invention may be prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for theimmunoglobulin genes of interest or a hybridoma prepared therefrom, (b)antibodies isolated from a host cell transformed to express the antibodyof interest, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of immunoglobulin gene sequences to other DNA sequences.

An antibody of the invention may be a human antibody or a humanisedantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which both the frameworkand CDR regions are derived from human germline immunoglobulinsequences. Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). However, the term “human antibody”, asused herein, is not intended to include antibodies in which CDRsequences derived from the germline of another mammalian species, suchas a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma that includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to CDR-graftedantibody molecules in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Additional framework region modifications may bemade within the human framework sequences.

As used herein, the term ‘CDR-grafted antibody molecule’ refers to anantibody molecule wherein the heavy and/or light chain contains one ormore CDRs (including, if desired, one or more modified CDRs) from adonor antibody (e.g. a murine or rat monoclonal antibody) grafted into aheavy and/or light chain variable region framework of an acceptorantibody (e.g. a human antibody). For a review, see Vaughan et al,Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather thanthe entire CDR being transferred, only one or more of the specificitydetermining residues from any one of the CDRs described herein above aretransferred to the human antibody framework (see for example, Kashmiriet al., 2005, Methods, 36, 25-34). In one embodiment only thespecificity determining residues from one or more of the CDRs describedherein above are transferred to the human antibody framework. In anotherembodiment only the specificity determining residues from each of theCDRs described herein above are transferred to the human antibodyframework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, primate and human framework regions.Suitably, the CDR-grafted antibody according to the present inventionhas a variable domain comprising human acceptor framework regions aswell as one or more of the CDRs or specificity determining residuesdescribed above. Thus, provided in one embodiment is a neutralisingCDR-grafted antibody wherein the variable domain comprises humanacceptor framework regions and non-human donor CDRs.

Examples of human frameworks which can be used in the present inventionare KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). Forexample, KOL and NEWM can be used for the heavy chain, REI can be usedfor the light chain and EU, LAY and POM can be used for both the heavychain and the light chain. Alternatively, human germline sequences maybe used; these are available for example at: http://www.vbase2.org/ (seeRetter et al, Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).

In a CDR-grafted antibody of the present invention, the acceptor heavyand light chains do not necessarily need to be derived from the sameantibody and may, if desired, comprise composite chains having frameworkregions derived from different chains.

Also, in a CDR-grafted antibody of the present invention, the frameworkregions need not have exactly the same sequence as those of the acceptorantibody. For instance, unusual residues may be changed to morefrequently occurring residues for that acceptor chain class or type.Alternatively, selected residues in the acceptor framework regions maybe changed so that they correspond to the residue found at the sameposition in the donor antibody (see Reichmann et al., 1998, Nature, 332,323-324). Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO 91/09967.

It will also be understood by one skilled in the art that antibodies mayundergo a variety of posttranslational modifications. The type andextent of these modifications often depends on the host cell line usedto express the antibody as well as the culture conditions. Suchmodifications may include variations in glycosylation, methionineoxidation, diketopiperazine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995).

In one embodiment the antibody heavy chain comprises a CH1 domain andthe antibody light chain comprises a CL domain, either kappa or lambda.

Biological molecules, such as antibodies or fragments, contain acidicand/or basic functional groups, thereby giving the molecule a netpositive or negative charge. The amount of overall “observed” chargewill depend on the absolute amino acid sequence of the entity, the localenvironment of the charged groups in the 3D structure and theenvironmental conditions of the molecule. The isoelectric point (pI) isthe pH at which a particular molecule or surface carries no netelectrical charge. In one embodiment the antibody or fragment accordingto the present disclosure has an isoelectric point (pI) of at least 7.In one embodiment the antibody or fragment has an isoelectric point ofat least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment the pI ofthe antibody is 8. Programs such as ** ExPASYhttp://www.expasy.ch/tools/pi_tool.html (see Walker, The ProteomicsProtocols Handbook, Humana Press (2005), 571-607), may be used topredict the isoelectric point of the antibody or fragment.

The antibody of the invention may comprise at least one, at least two orall three heavy chain CDR sequences of SEQ ID NOS: 4 to 6(HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3sequences of the CA185_01974 antibody of the Examples.

Furthermore, the antibody of the invention may comprise at least one, atleast two or all three light chain CDR sequences of SEQ ID NOS: 1 to 3(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3sequences of the CA185_01974 antibody of the Examples.

The antibody of the invention suitably comprises at least a HCDR3sequence of SEQ ID NO: 6.

Typically, the antibody of the invention comprises at least one heavychain CDR sequence selected from SEQ ID NOS: 4 to 6 and at least onelight chain CDR sequence selected from SEQ ID NOS 1 to 3. The antibodyof the invention may comprise at least two heavy chain CDR sequencesselected from SEQ ID NOS: 4 to 6 and at least two light chain CDRsequences selected from SEQ ID NOS: 1 to 3. The antibody of theinvention typically comprises all three heavy chain CDR sequences of SEQID NOS: 4 to 6 (HCDR1/HCDR2/HCDR3 respectively) and all three lightchain CDR sequences SEQ ID NOS: 1 to 3 (LCDR1/LCDR2/LCDR3 respectively).The antibodies may be chimeric, human or humanised antibodies.

The antibody of the invention may also comprise at least one, at leasttwo or all three heavy chain CDR sequences of SEQ ID NOS: 19 to 21(HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3sequences of the CA185_01979 antibody of the Examples.

The antibody typically comprises a HCDR3 sequence of SEQ ID NO: 21.

The antibody of the invention may also comprise at least one, at leasttwo or all three light chain CDR sequences of SEQ ID NOS: 1, 17, 18(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3sequences of the CA185_01979 antibody of the Examples

Typically, the antibody of the invention comprises at least one heavychain CDR sequence selected from SEQ ID NOS: 19 to 21 and at least onelight chain CDR sequence selected from SEQ ID NOS: 1, 17, 18. Theantibody of the invention may comprise at least two heavy chain CDRsequences selected from SEQ ID NOS: 19 to 21 and at least two lightchain CDR sequences selected from SEQ ID NOS: 1, 17, 18. The antibody ofthe invention typically comprises all three heavy chain CDR sequences ofSEQ ID NOS: 19 to 21 (HCDR1/HCDR2/HCDR3 respectively) and all threelight chain CDR sequences SEQ ID NOS: 1, 17, 18 (LCDR1/LCDR2/LCDR3respectively). The antibodies may be chimeric, human or humanisedantibodies.

The antibody of the invention may comprise any combination of CDRsequences of the CA185_01974 antibody and the CA185_01979 antibody. Inparticular, the antibody of the invention may comprise least one HCDRsequence selected from SEQ ID NOs: 4-6 and 19-21 and/or at least oneLCDR sequence selected from SEQ ID NOs: 1-3, 17 and 18.

The antibody may comprise:

-   -   a HCDR1 selected from SEQ ID NOs: 4 and 19; and/or    -   a HCDR2 selected from SEQ ID NOs: 5 and 20; and/or    -   a HCDR3 selected from SEQ ID NOs: 6 and 21; and/or    -   a LCDR1 of SEQ ID NO: 1; and/or    -   a LCDR2 selected from SEQ ID NOs: 2 and 17; and/or    -   a LCDR3 selected from SEQ ID NOs: 3 and 18.

The antibody of the invention may comprise a heavy chain variable region(HCVR) sequence of SEQ ID NO: 8 (the HCVR of CA185_01974). The antibodyof the invention may comprise a light chain variable region (LCVR)sequence of SEQ ID NO: 7 (the LCVR of CA185_01974). The antibody of theinvention suitably comprises the heavy chain variable region sequence ofSEQ ID NO: 8 and the light chain variable region sequence of SEQ ID NO:7.

The antibody of the invention may also comprise a heavy chain variableregion (HCVR) sequence of SEQ ID NO: 23 (the HCVR of CA185_01979). Theantibody of the invention may comprise a light chain variable region(LCVR) sequence of SEQ ID NO: 22 (the LCVR of CA185_01979). The antibodyof the invention suitably comprises the heavy chain variable regionsequence of SEQ ID NO: 23 and the light chain variable region sequenceof SEQ ID NO: 22.

Again, the antibody of the invention may comprise a combination of heavyand light chain variable regions from the CA185_01974 and CA185_01979antibodies. In other words, the antibody of the invention may comprise aheavy chain variable region of SEQ ID NO: 8 or 23 and/or a light chainvariable region of SEQ ID NO: 7 or 22.

The antibody of the invention may comprise a heavy chain (H-chain)sequence of SEQ ID NO: 12 (CA185_01974 mIgG1) or 13 (CA185_01974 mFab(no hinge)). The antibody of the invention may comprise a light chain(L-chain) sequence of SEQ ID NO: 11 (CA185_01974 kappa light chain). Theantibody of the invention typically comprises the heavy chain sequenceof SEQ ID NO: 12/13 and the light chain sequence of SEQ ID NO: 11. Theantibodies may be chimeric, human or humanised antibodies.

The antibody of the invention may comprise a heavy chain sequence of SEQID NO: 27 (CA185_01979 mIgG1) or 28 (CA185_01979 mFab (no hinge)). Theantibody of the invention may comprise a light chain sequence of SEQ IDNO: 26 (CA185_01979 kappa light chain). Generally, the antibody of theinvention comprises the heavy chain sequence of SEQ ID NO: 27/28 and thelight chain sequence of SEQ ID NO: 26. The antibodies may be chimeric,human or humanised antibodies. Again, sequences from CA185_01974 andCA185_01979 may be combined.

The antibody may alternatively be or may comprise a variant of one ofthe specific sequences recited above. For example, a variant may be asubstitution, deletion or addition variant of any of the above aminoacid sequences.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20 ormore (typically up to a maximum of 50) amino acid substitutions and/ordeletions from the specific sequences discussed above. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Substitution” variants typicallyinvolve the replacement of one or more amino acids with the same numberof amino acids and making conservative amino acid substitutions. Forexample, an amino acid may be substituted with an alternative amino acidhaving similar properties, for example, another basic amino acid,another acidic amino acid, another neutral amino acid, another chargedamino acid, another hydrophilic amino acid, another hydrophobic aminoacid, another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as follows:

Ala aliphatic, hydrophobic, Met hydrophobic, neutral neutral Cys polar,hydrophobic, Asn polar, hydrophilic, neutral neutral Asp polar,hydrophilic, Pro hydrophobic, neutral charged (−) Glu polar,hydrophilic, Gln polar, hydrophilic, charged (−) neutral Phe aromatic,hydrophobic, Arg polar, hydrophilic, neutral charged (+) Gly aliphatic,neutral Ser polar, hydrophilic, neutral His aromatic, polar, Thr polar,hydrophilic, hydrophilic, neutral charged (+) Ile aliphatic,hydrophobic, Val aliphatic, hydrophobic, neutral neutral Lys polar,hydrophilic, Trp aromatic, hydrophobic, charged (+) neutral Leualiphatic, hydrophobic, Tyr aromatic, polar, neutral hydrophobic

“Derivatives” or “variants” generally include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatized or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post-production modification, or whenthe antibody is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

Variant antibodies may have an amino acid sequence which has more thanabout 60%, or more than about 70%, e.g. 75 or 80%, preferably more thanabout 85%, e.g. more than about 90 or 95% amino acid identity to theamino acid sequences disclosed herein (particularly the HCVR/LCVRsequences and the H- and L-chain sequences). Furthermore, the antibodymay be a variant which has more than about 60%, or more than about 70%,e.g. about 75 or 80%, typically more than about 85%, e.g. more thanabout 90 or 95% amino acid identity to the HCVR/LCVR sequences and theH- and L-chain sequences disclosed herein, whilst retaining the exactCDRs disclosed for these sequences. Variants may retain at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to theHCVR/LCVR sequences and to the H- and L-chain sequences disclosed herein(in some circumstances whilst retaining the exact CDRs).

Variants typically retain about 60%-about 99% identity, about 80%-about99% identity, about 90%-about 99% identity or about 95%-about 99%identity. This level of amino acid identity may be seen across the fulllength of the relevant SEQ ID NO sequence or over a part of thesequence, such as across about 20, 30, 50, 75, 100, 150, 200 or moreamino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatized.

The present invention thus provides antibodies having specific sequencesand variants which maintain the function or activity of these chains.

The present invention also provides an isolated DNA sequence encodingthe heavy and/or light chain variable regions(s) of an antibody moleculeof the present invention. Thus, the present invention provides anisolated DNA sequence of SEQ ID NO: 10, which encodes the heavy chainvariable region of SEQ ID NO: 8. The invention also provides an isolatedDNA sequence of SEQ ID NO: 9, which encodes the light chain variableregion of SEQ ID NO: 7.

The present invention also provides an isolated DNA sequence of SEQ IDNO: 25, which encodes the heavy chain variable region of SEQ ID NO: 23.The invention also provides an isolated DNA sequence of SEQ ID NO: 24,which encodes the light chain variable region of SEQ ID NO: 22.

The present invention also provides an isolated DNA sequence encodingthe heavy and/or light chain(s) of an antibody molecule of the presentinvention. Suitably, the DNA sequence encodes the heavy or the lightchain of an antibody molecule of the present invention. Thus, thepresent invention provides an isolated DNA sequence of SEQ ID NO: 15 or16, which encode the heavy chains of SEQ ID NOs: 12 and 13 respectively.The invention also provides an isolated DNA sequence of SEQ ID NO: 14,which encodes the light chain of SEQ ID NO: 11.

The present invention also provides an isolated DNA sequence of SEQ IDNO: 30 or 31, which encode the heavy chains of SEQ ID NOs: 27 and 28respectively. The invention also provides an isolated DNA sequence ofSEQ ID NO: 29, which encodes the light chain of SEQ ID NO: 26.

A suitable polynucleotide sequence may alternatively be a variant of oneof these specific polynucleotide sequences. For example, a variant maybe a substitution, deletion or addition variant of any of the abovenucleic acid sequences. A variant polynucleotide may comprise 1, 2, 3,4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or morenucleic acid substitutions and/or deletions from the sequences given inthe sequence listing. Generally, a variant has 1-20, 1-50, 1-75 or 1-100substitutions and/or deletions.

Suitable variants may be at least about 70% homologous to apolynucleotide of any one of nucleic acid sequences disclosed herein,typically at least about 80 or 90% and more suitably at least about 95%,97% or 99% homologous thereto. Variants may retain at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Variantstypically retain about 60%-about 99% identity, about 80%-about 99%identity, about 90%-about 99% identity or about 95%-about 99% identity.Homology and identity at these levels is generally present at least withrespect to the coding regions of the polynucleotides. Methods ofmeasuring homology are well known in the art and it will be understoodby those of skill in the art that in the present context, homology iscalculated on the basis of nucleic acid identity. Such homology mayexist over a region of at least about 15, at least about 30, forinstance at least about 40, 60, 100, 200 or more contiguous nucleotides(depending on the length). Such homology may exist over the entirelength of the unmodified polynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al (1984) Nucleic Acids Research 12, p 387-395).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul,S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4,and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, typically less than about 0.1, suitably less thanabout 0.01, and most suitably less than about 0.001. For example, thesmallest sum probability may be in the range of about 1-about 0.001,often about 0.01-about 0.001.

The homologue may differ from a sequence in the relevant polynucleotideby less than about 3, 5, 10, 15, 20 or more mutations (each of which maybe a substitution, deletion or insertion). For example, the homologuemay differ by 3-50 mutations, often 3-20 mutations. These mutations maybe measured over a region of at least 30, for instance at least about40, 60 or 100 or more contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specificsequences given in the sequence listing by virtue of the redundancy inthe genetic code. The DNA code has 4 primary nucleic acid residues (A,T, C and G) and uses these to “spell” three letter codons whichrepresent the amino acids the proteins encoded in an organism's genes.The linear sequence of codons along the DNA molecule is translated intothe linear sequence of amino acids in the protein(s) encoded by thosegenes. The code is highly degenerate, with 61 codons coding for the 20natural amino acids and 3 codons representing “stop” signals. Thus, mostamino acids are coded for by more than one codon—in fact several arecoded for by four or more different codons. A variant polynucleotide ofthe invention may therefore encode the same polypeptide sequence asanother polynucleotide of the invention, but may have a differentnucleic acid sequence due to the use of different codons to encode thesame amino acids.

The DNA sequence of the present invention may comprise synthetic DNA,for instance produced by chemical processing, cDNA, genomic DNA or anycombination thereof.

DNA sequences which encode an antibody molecule of the present inventioncan be obtained by methods well known to those skilled in the art. Forexample, DNA sequences coding for part or all of the antibody heavy andlight chains may be synthesised as desired from the determined DNAsequences or on the basis of the corresponding amino acid sequences.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present invention. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present invention. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

Screening methods as described herein may be used to identify suitableantibodies that are capable of binding to a compound-trimer complex.Thus, the screening methods described herein may be carried out to testantibodies of interest.

Antibodies of the invention can be tested for binding to acompound-trimer complex by, for example, standard ELISA or Westernblotting. An ELISA assay can also be used to screen for hybridomas thatshow positive reactivity with the target protein. The bindingselectivity of an antibody may also be determined by monitoring bindingof the antibody to cells expressing the target protein, for example byflow cytometry. Thus, a screening method of the invention may comprisethe step of identifying an antibody that is capable of binding acompound-trimer complex by carrying out an ELISA or Western blot or byflow cytometry.

Antibodies of the invention selectively (or specifically) recognise atleast one compound-trimer complex, i.e. epitopes within acompound-trimer complex. An antibody, or other compound, “selectivelybinds” or “selectively recognises” a protein when it binds withpreferential or high affinity to the protein for which it is selectivebut does not substantially bind, or binds with low affinity, to otherproteins. The selectivity of an antibody of the invention for a target acompound-trimer complex may be further studied by determining whether ornot the antibody binds to other related compound-trimer complexes asdiscussed above or whether it discriminates between them.

An antibody of the invention may bind specifically (or selectively) tocompound-trimer complexes comprising the trimeric forms of one or moreTNF superfamily members. For example, an antibody may bind tocompound-trimer complexes comprising TNFα, compound-trimer complexescomprising TNFβ and compound-trimer complexes comprising CD40L.Alternatively, an antibody may bind specifically (or selectively) tocompound-trimer complexes comprising only one of the TNF superfamilymembers, but not to compound-trimer complexes comprising any other TNFsuperfamily members. For example, an antibody may bind tocompound-trimer complexes comprising TNFα, but not to compound-trimercomplexes comprising TNFβ or compound-trimer complexes comprising CD40L.An antibody may bind specifically (or selectively) to compound-trimercomplexes comprising up to two, three, four or up to all of the TNFsuperfamily members.

By specific (or selective), it will be understood that the antibodybinds to the compound-trimer complexes of interest with no significantcross-reactivity to any other molecule, which may include test compoundsin the absence of a TNF superfamily trimer or TNF superfamily membertrimers in the absence of a test compound. Cross-reactivity may beassessed by any suitable method described herein. Cross-reactivity of anantibody for a compound-trimer complex with a molecule other than thecompound-trimer complex may be considered significant if the antibodybinds to the other molecule at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% asstrongly as it binds to the compound-trimer complex of interest. Anantibody that is specific (or selective) for the compound-trimer complexmay bind to another molecule at less than about 90%, 85%, 80%, 75%, 70%,65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that itbinds to the compound-trimer complex. The antibody may bind to the othermolecule at less than about 20%, less than about 15%, less than about10% or less than about 5%, less than about 2% or less than about 1% thestrength that it binds to the compound-trimer complex. The antibodyspecifically (or selectively) binds to a compound-trimer complexcompared with (i) the trimeric form of the TNF superfamily member in theabsence of the compound and/or (ii) the compound in the absence of theTNF superfamily member trimer.

The rates at which an antibody binds to a compound-trimer complex isreferred to herein as the “on” rate” k_(on-ab) and the rate at which theantibody dissociates from the compound-trimer complex is referred toherein as the “off” rate or k_(off-ab). As used herein, the symbol“K_(D-ab)” denotes the binding affinity (dissociation constant) of anantibody for a compound-trimer complex. K_(D-ab) is defined ask_(off-ab)/k_(on-ab). Antibodies may have slow “on” rates, which can bemeasured in minutes by mass spectral analysis of the compound-trimercomplex and antibody peak intensities. K_(D-ab) values for an antibodycan be estimated by repeating this measurement at different antibody:compound-trimer complex ratios.

The K_(D-ab) value of the antibody for binding to a compound-trimercomplex may be at least about 1.5 times, 2 times, 3 times, 4 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times, 200 times, 300 times or 400 timeslower, or lower, than the K_(D-ab) value of the antibody for binding tothe trimeric TNF superfamily member in the absence of the compoundand/or the K_(D-ab) value of the antibody for binding to the compound inthe absence of the trimeric TNF superfamily member. The K_(D-ab) valueof the antibody for binding to a compound-trimer complex may bedecreased at least about 10 times, at least about 100 times, at leastabout 200 times, at least about 300 times the K_(D-ab) value of the TNFsuperfamily trimer binding to the TNF superfamily receptor in theabsence of the test compound, i.e. the binding affinity of the antibodyfor the compound-trimer complex is typically increased at least about10-fold, suitably at least about 100-fold, more suitably at least about200-fold, most suitably at least about 300-fold compared to the bindingaffinity of the antibody to the trimeric TNF superfamily member in theabsence of the compound and/or the binding affinity of the antibody tothe compound in the absence of the trimeric TNF superfamily member.

The binding affinity may be given in terms of binding affinities(K_(D-ab)) and may be given in any appropriate units, such as μM, nM orpM. The smaller the K_(D-ab) value, the larger the binding affinity ofthe antibody to the compound-trimer complex.

The K_(D-ab) value of the antibody for binding to the compound-trimercomplex may be at least about 1.5 times, 2 times, 3 times, 4 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times lower, or even lower than theK_(D-ab) value of the antibody for binding to the trimeric TNFsuperfamily member in the absence of the compound and/or the K_(D-ab)value of the antibody for binding to the compound in the absence of thetrimeric TNF superfamily member.

The decrease in the K_(D-ab) value of the antibody for binding to thecompound-trimer complex compared to the K_(D-ab) value of the antibodybinding to the trimeric TNF superfamily member in the absence of thecompound and/or the K_(D-ab) value of the antibody for binding to thecompound in the absence of the trimeric TNF superfamily member mayresult from an increase in the on rate (k_(on-ab)) of the antibodybinding to the compound-trimer complex compared to the antibody bindingto the trimeric TNF superfamily member in the absence of the compoundand/or the antibody binding to the compound in the absence of thetrimeric TNF superfamily member; and/or a decrease in the off rate(k_(off-ab)) compared to the antibody binding to the trimeric TNFsuperfamily member in the absence of the compound and/or the antibodybinding to the compound in the absence of the trimeric TNF superfamilymember.

The on rate (k_(on-ab)) of the antibody binding to the compound-trimercomplex is generally increased compared to the on rate of the antibodybinding to the trimeric TNF superfamily member in the absence of thecompound and/or the antibody binding to the compound in the absence ofthe trimeric TNF superfamily member. The off rate (k_(off-ab)) of theantibody binding to the compound-trimer complex is generally decreasedcompared to the off rate of the antibody binding to the trimeric TNFsuperfamily member in the absence of the compound and/or the antibodybinding to the compound in the absence of the trimeric TNF superfamilymember. Most typically, the on rate (k_(on-ab)) of the antibody bindingto the compound-trimer complex is increased, and the off-rate(k_(off-ab)) of the antibody binding to the compound-trimer complex isdecreased, compared to the antibody binding to the trimeric TNFsuperfamily member in the absence of the compound and/or the antibodybinding to the compound in the absence of the trimeric TNF superfamilymember.

The k_(on-ab) value of the antibody binding to the compound-trimercomplex may be increased by at least about 1.5-fold or at least two-foldand typically at least about three fold compared to the k_(on-ab) valueof the antibody binding to the trimeric TNF superfamily member in theabsence of the compound and/or the antibody binding to the compound inthe absence of the trimeric TNF superfamily member and/or the k_(off-ab)value of the antibody binding to the compound-trimer complex may bedecreased by at least about two-fold, at least about 10-fold, at leastabout 20-fold, at least about 30-fold, at least about 40-fold, at leastabout 50-fold, at least about 60-fold, at least about 70-fold, at leastabout 80-fold more suitably at least about 90-fold compared to thek_(off-ab) value of the antibody binding to to the trimeric TNFsuperfamily member in the absence of the compound and/or the antibodybinding to the compound in the absence of the trimeric TNF superfamilymember.

The k_(on-ab), k_(off-ab), and K_(D-ab) values may be determined usingany appropriate technique, for example surface plasmon resonance, massspectrometry and isothermal calorimetry.

The K_(D-ab) value of the antibody binding to a compound-trimer complexmay be 1 nM, 900 pM, 700 pM, 500 pM, 100 pM, 10 pM or less (typicallydown to about 1 pM). Antibodies of the invention will desirably bind tothe compound-trimer complexes of the invention with high affinity, forexample in the picomolar range. The K_(D-ab) value of the antibodybinding to a compound-trimer complex may be 1 nM or less, 900 pM orless, 700 pM or less, 500 pM or less, 400 pM or less, 300 pM or less,200 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM orless, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pMor less, 10 pM or less (again, down to about 1 pM).

Once a suitable antibody has been identified and selected, the aminoacid sequence of the antibody may be identified by methods known in theart. The genes encoding the antibody can be cloned using degenerateprimers. The antibody may be recombinantly produced by routine methods.

Antibodies of the invention may compete for binding to TNFα with, orbind to the same epitope as, those defined above in terms ofH-chain/L-chain, HCVR/LCVR or CDR sequences. In particular, an antibodymay compete for binding to TNFα with, or bind to the same epitope as, anantibody which comprises a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequencecombination of SEQ ID NOs: 4/5/6/1/2/3 or SEQ ID NOs: 19/20/21/1/17/18.An antibody may compete for binding to TNFα with, or bind to the sameepitope as, an antibody which comprises a HCVR and LCVR sequence pair ofSEQ ID NOs: 8/7 or SEQ ID NOs: 23/22.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference antibody by using routinemethods known in the art. For example, to determine if a test antibodybinds to the same epitope as a reference antibody of the invention, thereference antibody is allowed to bind to a protein or peptide undersaturating conditions. Next, the ability of a test antibody to bind tothe protein or peptide is assessed. If the test antibody is able to bindto the protein or peptide following saturation binding with thereference antibody, it can be concluded that the test antibody binds toa different epitope than the reference antibody. On the other hand, ifthe test antibody is not able to bind to protein or peptide followingsaturation binding with the reference antibody, then the test antibodymay bind to the same epitope as the epitope bound by the referenceantibody of the invention.

To determine if an antibody competes for binding with a referenceantibody, the above-described binding methodology is performed in twoorientations. In a first orientation, the reference antibody is allowedto bind to a protein/peptide under saturating conditions followed byassessment of binding of the test antibody to the protein/peptidemolecule. In a second orientation, the test antibody is allowed to bindto the protein/peptide under saturating conditions followed byassessment of binding of the reference antibody to the protein/peptide.If, in both orientations, only the first (saturating) antibody iscapable of binding to the protein/peptide, then it is concluded that thetest antibody and the reference antibody compete for binding to theprotein/peptide. As will be appreciated by the skilled person, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50%, 75%, 90% or even 99% as measuredin a competitive binding assay (see, e.g., Junghans et al., Cancer Res,1990:50:1495-1502). Alternatively, two antibodies have the same epitopeif essentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies have overlapping epitopes if some amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

The antibodies of the invention may be used to identify compounds of theinvention as described herein. The antibodies of the invention may alsobe used as target engagement biomarkers. A target engagement biomarkercan be used to detect the engagement, i.e. the binding of a ligand to atarget of interest. In the present case, the antibodies of the inventiononly bind to the complexes of compounds of the invention with trimericforms of TNF superfamily members. Therefore, if an antibody of theinvention is able to bind to a compound-trimer complex, this is evidencethat the ligand (compound) has bound to the target of interest (TNFsuperfamily member trimer). Antibodies of the invention can be modifiedto add a detectable marker as described herein. Therefore, engagement ofa compound of the invention with a target TNF superfamily member may bedetected using such an antibody.

The use of antibodies of the invention as target engagement biomarkersis potentially useful in a clinical or pre-clinical environment, where asample may be taken from a subject being treated according to thepresent invention. The sample obtained from the subject may be treatedwith an antibody of the invention in order to determine whether thecompound used to treat the subject has bound to the target TNFsuperfamily member. The sample obtained from the subject may be anyappropriate tissue or fluid, such as blood, plasma or urine. The subjectmay be mammalian, typically human.

Accordingly, the invention provides the use of an antibody of theinvention as a target engagement biomarker for the detection of acompound-trimer complex comprising a trimeric protein that is a TNFsuperfamily member and a compound that is capable of binding to atrimeric protein that is a TNF superfamily member, whereby thecompound-trimer complex binds to the requisite TNF superfamily receptorand modulates the signalling induced by the trimer through the receptorin a sample obtained from a subject. The superfamily member is suitablyTNFα and/or the modulation is antagonism of TNFR1 signalling.

Similarly, the present invention provides a method of detecting targetengagement of a compound to a trimeric TNF superfamily member, wherebythe compound-trimer complex binds to the requisite receptor andmodulates the signalling induced by the trimer through the receptor,said method comprising:

(a) obtaining a sample from a subject administered said compound;

(b) contacting an antibody of the invention to said sample and a controlsample, wherein said antibody is detectable;

(c) determining the amount of binding of said detectable antibody tosaid sample and said control sample,

wherein binding of said detectable antibody to said sample greater thanbinding of said detectable antibody to said control sample indicatestarget engagement of said compound to said trimeric TNF superfamilymember.

Methods of detecting antibodies, and measuring the amount of binding ofan antibody to a target, are well known in the art. Typically,antibodies can be labelled. Such labels include enzymes,biotin/streptavidin, fluorescent proteins and fluorescent dyes.

Binding of an antibody to a target may be measured, for example, by animmunoassay method. Immunoassays include Western Blotting, ELISA,immunofluorescence, immunohistochemistry and flow cytometry. Anyappropriate technique may be used to measure binding of the antibody tothe TNF superfamily member.

In the method described above, binding of the detectable antibody to thesample from a subject who has been administered the compound is comparedwith binding of the antibody to a control sample. The control sample maybe any appropriate sample. The control sample is typically a “negativecontrol” which is representative of binding of the antibody to the TNFsuperfamily member in the absence of the compound. For example, thesample may be obtained from the patient prior to administration of thecompound. The control may also be based on previously determinedmeasurements e.g. from a number of samples from different subjects inthe absence of compound. Measurements from about 5, 10, 20, 50 or 100subjects may be used in determining the control value. The control maybe an average value, or a range of all the values obtained.

The experimental conditions e.g. methods of detection are the same forthe sample from a subject administered the compound, and for the controlsample. The antibody is also the same in both cases.

Greater binding (increased binding) of the detectable antibody to thesample from the patient administered the compound compared with bindingof the antibody to the control sample is indicative of target engagementof the compound to the trimeic TNF superfamily member. In other words,equivalent or lower binding (decreased binding) for the sample from thepatient administered the compound relative to the control indicates thatthere is no target engagement of said compound. In other words, nosignificant difference in the two amounts indicates that there is notarget engagement.

The skilled person can readily determine when there is increased bindingrelative to the control. For example when the control is a range ofdata, target engagement may be determined based on the spread of thedata, the difference between the control data and the detected bindingof the antibody in the sample in question, and calculated confidencelevels. It is also possible to identify target engagement when thedetected binding for the sample in question is higher than the maximumamount of binding detected in any negative control.

Target engagement may be detected if binding of the antibody isincreased by about 30% or more relative to the highest amount in thecontrol range. Target engagement may also be detected if binding of theantibody is increased by about 40% or more, or about 50% or morerelative to the control range. The same applies when the control is anaverage value, or a single value based on a sample from the patientprior to administration of the compound. There is of course no upperlimit to the percentage increase relative to the control.

An antibody of the invention may be used to screen for a compound thatelicits a conformational change in a trimeric TNF superfamily member,wherein said conformational change modulates the signalling of therequisite TNF superfamily receptor on binding of the trimeric TNFsuperfamily member. The superfamily member is typically TNFα and/or themodulation is antagonism of TNFR1 signalling.

The antibodies of the present invention may be used in the treatmentand/or prophylaxis of a pathological condition. Accordingly, provided isan antibody of the invention for use in a method of therapy practiced onthe human or animal body. The invention also provides a method oftherapy comprising the administration of an antibody of the invention toa subject. The antibody of the invention may be used in any therapeuticindication and/or pharmaceutical composition described herein.

Antibody Assays

As described herein, the present invention provides antibodies thatselectively bind to at least one compound-trimer complex describedherein relative to their binding to the compound alone or to the TNFsuperfamily member in the absence of the compound. These antibodies maybe used to identify further compounds or classes of compounds having thesame properties.

Monoclonal antibodies may be generated against a TNF superfamily memberusing the standard techniques described herein. These anti-TNFsuperfamily member antibodies can then be screened for antibodies thatbind to compound-trimer complexes of the invention, or for monoclonalantibodies for which binding to the TNF superfamily member is inhibitedby compounds as described herein.

Alternatively, monoclonal antibodies can be generated against particularTNF superfamily member trimer-compound complexes. These antibodies canthen be screened for monoclonal antibodies that selectively bind to theTNF superfamily member in the presence of the compound relative to theirbinding to the TNF superfamily member in the absence of the compound.

Once an antibody that selectively binds to at least one compound-trimercomplex of the invention relative to its binding to the compound aloneor to the TNF superfamily member in the absence of the compound has beengenerated, it can be used to screen for other compounds possessing thesame activity as the test compounds.

Accordingly, the invention provides an assay for identifying a compoundof the invention comprising the steps of:

a) performing a binding assay to measure the binding affinity of a testcompound-trimer complex to an antibody of the invention;b) comparing the binding affinity as measured in step (a) with thebinding affinity of a different compound-trimer complex known to bindwith high affinity to the antibody referred to in step (a); andc) selecting the compound present in the compound-trimer complex of step(a) if its measured binding affinity is acceptable when considered inthe light of the comparison referred to in step (b).

As will be appreciated, the “different” compound-trimer complex referredto in step (b) above will generally be a complex containing the sametrimer as the compound-trimer complex of step (a), but a differentcompound. The compound may be any of compounds (1)-(6).

By “acceptable” in step (c) is meant that the binding affinity of thecompound-trimer complex referred to in step (a) and the binding affinityof the different compound-trimer complex referred to in step (b) areapproximately comparable. Selective binding of said antibody to saidcomplex is typically measured relative to the binding of said antibodyto the TNF superfamily member in the absence of the compound or to thecompound in the absence of the TNF superfamily member.

The binding affinity of the compound-trimer complex referred to in step(a) will generally be superior to the binding affinity of the differentcompound-trimer complex referred to in step (b). Suitably, thedifference in the binding affinity of the compound-trimer complexreferred to in step (a) relative to the binding affinity of thedifferent compound-trimer complex referred to in step (b) will be withinlimits of 10-fold, 20-fold, 50-fold, 100-fold, 200-fold or 500-fold.

Libraries of compounds can be assayed using the antibodies of theinvention. The library compounds can be incubated with said antibody inthe presence and absence of a TNF superfamily member. A compound thatforms part of a compound-trimer complex that binds to an antibody of theinvention only in the presence of both the TNF superfamily member andthe compound is a likely candidate to have the same activity as thecompounds described herein. The assays disclosed herein may then be usedto verify whether the test compound is a compound as described herein.

One or more of the antibodies of the invention may be used in the assay.A generic antibody that is capable of binding to complexes of anycompound of the invention with a particular TNF superfamily member maybe used in the antibody assay of the invention.

A panel of multiple antibodies of the present invention that arespecific for different compound-trimer complexes may be used in theantibody assay of the invention. The panel of antibodies may include atleast 5, at least 10, at least 15, at least 20, at least 30, at least 40or at least 50 antibodies (for example up to 75 antibodies).

The antibody assay of the present invention may be a high throughputassay that is capable of screening a large number of test compounds overa short space of time to identify compounds of the present invention.

The TNF superfamily members and their receptors may be purified orpresent in mixtures, such as in cultured cells, tissue samples, bodyfluids or culture medium. Assays may be developed that are qualitativeor quantitative, with the latter being useful for determining thebinding parameters (affinity constants and kinetics) of the testcompound to trimeric forms of TNF superfamily members, and also of thebinding parameters of the compound-trimer complex to the requisite TNFreceptor.

The sample comprising the TNF superfamily member and the compound mayfurther comprise a destabilising agent. Destabilising agents, also knownas chaotropes, include low molar concentrations (e.g. 1M) of urea,guanidine or acetonitrile, high concentrations (e.g. 6M or higher) ofthese reagents will result in complete dissociation of the TNFα trimerand unfolding of the constituent TNFα monomeric subunits. Thedestabilising agent may be DMSO, typically at a concentration of 5%, 10%or higher.

The test compounds may have any/all of the properties discussed above.

TNF Superfamily and their Receptors

There are 22 TNF superfamily members currently known: TNFα (TNFSF1A),TNFβ (TNFSF1B), CD40L (TNFSF5), BAFF (TNFSF13B/BlyS), APRIL (TNFSF13),OX40L (TNFSF4), RANKL (TNFSF11/TRANCE), TWEAK (TNFSF12), TRAIL(TNFSF10), TL1A (TNFSF15), LIGHT (TNFSF14), Lymphotoxin, Lymphotoxin β(TNFSF3), 4-1BBL (TNFSF9), CD27L (TNFSF7), CD03L (TNFSF8), EDA(Ectodysplasin), EDA-A1 (Ectodysplasin A1), EDA-A2 (Ectodysplasin A2),FASL (TNFSF6), NGF and GITRL (TNFSF18).

The TNF superfamily member is typically TNFα. TNFα exists in both asoluble (TNFα_(s)) and membrane-bound form (TNFα_(m)). When TNFα isreferred to herein this encompasses both the TNFα_(s) and TNFα_(m)forms. TNFα is most suitably in the TNFα_(s) form. The TNFα_(s) maycomprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36, or a variantthereof (as described above).

The assays of the invention may be used to identify modulators of atleast one of any TNF superfamily members, including the 22 known TNFsuperfamily members. Specifically, the assays of the invention may beused to identify compounds that bind to any TNF superfamily member,particularly to trimeric forms of TNF superfamily members, and thatstabilise these trimers in a conformation that is capable of binding tothe requisite TNF receptor, and which modulate signalling through saidreceptor. The assay of the invention is, in particular, used to identifymodulators of TNFα or CD40L, especially TNFα, or even TNFα_(s).

The compound described herein may be a modulator of at least one of anyTNF superfamily members, including the 22 known TNF superfamily members.In particular, the TNF superfamily member is TNFα or CD40L, especiallyTNFα or even TNFα_(s).

The compound-trimer complex of the invention may include the trimericform of any TNF superfamily member, including the 22 known TNFsuperfamily members. The TNF superfamily member is typically TNFα orCD40L. The TNF superfamily member may be TNFα, most suitably TNFα_(s).

Members of the TNF superfamily bind to, and initiate signalling throughTNF receptors. There are currently 34 known TNF receptors: 4-1BB(TNFRSF9/CD137), NGF R (TNFRSF16), BAFF R (TNFRSF13C), Osteoprotegerin(TNFRSF11B), BCMA (TNFRSF17), OX40 (TNFRSF4), CD27 (TNFRSF7), RANK(TNFRSF11A), CD30 (TNFRSF8), RELT (TNFRSF19L), CD40 (TNFRSF5), TACI(TNFRSF13B), DcR3 (TNFRSF6B), TNFRH3 (TNFRSF26), DcTRAIL R1 (TNFRSF23),DcTRAIL R2 (TNFRSF22), TNF-R1 (TNFRSF1A), TNF-R2 (TNFRSF1B), DR3(TNFRSF25), TRAIL R1 (TNFRSF10A), DR6 (TNFRSF21), TRAIL R2 (TNFRSF10B),EDAR, TRAIL R3 (TNFRSF10C), Fas (TNFRSF6/CD95), TRAIL R4 (TNFRSF10D),GITR (TNFRSF18), TROY (TNFRSF19), HVEM (TNFRSF14), TWEAK R (TNFRSF12A),TRAMP (TNFRSF25), Lymphotoxin β R (TNFRSF3) and XEDAR.

The TNF receptor is suitably TNF-R1 (TNFR1) or TNF-R2 (TNFR2). WhenTNF-R is referred to herein this encompasses both TNF-R1 and TNF-R2,including the extracellular domain (ECD) of TNF-R1 and TNF-R2. Theassays of the invention may be used to identify compounds that modulatethe signalling of TNF superfamily members through any requisite TNFsuperfamily receptor. The assays of the invention may be used toidentify compounds that modulate the signalling of TNF superfamilymembers through TNF-R1, TNF-R2 or CD40. The TNF superfamily member maybe TNFα and the TNF receptor may be TNF-R1 or TNF-R2. In particular, theTNF superfamily member may be TNFα and the TNF receptor may be TNF-R1.More particularly, the TNF superfamily member may be TNFα_(s) and theTNF receptor may be TNF-R1. The assays of the invention may be used toidentify compounds which act by specifically modulate the signalling ofTNF superfamily members through TNF-R1. In particular, the compounds mayact by modulating the signalling of TNF superfamily members throughTNF-R1, but have no effect on signalling of TNF superfamily membersthrough TNF-R2.

The compound-trimer complex of the invention may modulate TNFsuperfamily members signalling through at least one TNF receptor,including the 34 known TNF receptors. The TNF receptor is typicallyTNF-R1, TNF-R2 or CD40L.

In particular, the TNF superfamily member is TNFα and the TNF receptoris TNF-R1 or TNF-R2. The TNF superfamily member is more suitably TNFαand the TNF receptor is TNF-R1. Most suitably, the TNF superfamilymember is TNFα_(s) and the TNF receptor is TNF-R1.

Therapeutic Indications

TNFα is the archetypal member of the TNF superfamily. TNFα is apleiotropic cytokine that mediates immune regulation and inflammatoryresponses. In vivo, TNFα is also known to be involved in responses tobacterial, parasitic and viral infections. In particular, TNFα is knownto have a role in rheumatoid arthritis (RA), inflammatory bowel diseases(including Crohn's disease), psoriasis, Alzheimer's disease (AD),Parkinson's disease (PD), pain, epilepsy, osteoporosis, asthma, sepsis,fever, Systemic lupus erythematosus (SLE) and Multiple Sclerosis (MS)and cancer. TNFα is also known to have a role in Amyotrophic LateralSclerosis (ALS), ischemic stroke, immune complex-mediatedglomerulonephritis, lupus nephritis (LN), antineutrophil cytoplasmicantibodies (ANCA-) associated glomerulonephritis, minimal changedisease, diabetic nephropathy (DN), acute kidney injury (AKI),obstructive uropathy, kidney allograft rejection, cisplatin-induced AKIand obstructive uropathy.

Other members of the TNF superfamily are known to be involved inautoimmune disease and immune deficiencies. In particular, members ofthe TNF superfamily are known to be involved in RA, SLE, cancer, MS,asthma, rhinitis, osteoporosis and multiple myeloma (MM). TL1A is knownto play a role in organ transplant rejection.

A compound described herein may be used to treat, prevent or ameliorateany condition that that can be treated, prevented or ameliorated by aconventional TNF superfamily member modulator. The compound may be usedalone or in combination with a conventional TNF superfamily membermodulator. Any condition that results, partially or wholly, frompathogenic signalling through a TNF receptor by a TNF superfamily memberor from a deficiency in signalling through a TNF receptor by a TNFsuperfamily member may in principle be treated, prevented or amelioratedaccording to the present invention. Pathogenic signalling through a TNFreceptor by a TNF superfamily member includes increased signallingthrough a TNF receptor over and above the normal physiological level ofsignalling, signalling through a TNF receptor which is initiatednormally, but which fails to stop in response to normal physiologicalsignals and signalling through a TNF receptor that is within the normalphysiological range of magnitude, but which is initiated bynon-physiological means. In a preferred embodiment, the inventionrelates to the treatment, prevention or amelioration of conditionsmediated or influenced by TNFα or CD40L.

The compounds that interact with TNFα are accordingly beneficial in thetreatment and/or prevention of various human ailments. These includeautoimmune and inflammatory disorders; neurological andneurodegenerative disorders; pain and nociceptive disorders; andcardiovascular disorders.

Inflammatory and autoimmune disorders include systemic autoimmunedisorders, autoimmune endocrine disorders and organ-specific autoimmunedisorders. Systemic autoimmune disorders include systemic lupuserythematosus (SLE), psoriasis, vasculitis, polymyositis, scleroderma,multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis andSjögren's syndrome. Autoimmune endocrine disorders include thyroiditis.Organ-specific autoimmune disorders include Addison's disease,haemolytic or pernicious anaemia, glomerulonephritis (includingGoodpasture's syndrome), Graves' disease, idiopathic thrombocytopenicpurpura, insulin-dependent diabetes mellitus, juvenile diabetes,uveitis, inflammatory bowel disease (including Crohn's disease andulcerative colitis), pemphigus, atopic dermatitis, autoimmune hepatitis,primary biliary cirrhosis, autoimmune pneumonitis, autoimmune carditis,myasthenia gravis, spontaneous infertility, osteoporosis, asthma andmuscular dystrophy (including Duchenne muscular dystrophy).

Neurological and neurodegenerative disorders include Alzheimer'sdisease, Parkinson's disease, Huntington's disease, stroke, amyotrophiclateral sclerosis, spinal cord injury, head trauma, seizures andepilepsy.

Cardiovascular disorders include thrombosis, cardiac hypertrophy,hypertension, irregular contractility of the heart (e.g. during heartfailure), and sexual disorders (including erectile dysfunction andfemale sexual dysfunction).

In particular, a compound may be used to treat or prevent inflammatorydisorders, CNS disorders, immune disorders and autoimmune diseases,pain, osteoporosis, fever and organ transplant rejection. A compound maybe used to treat or prevent rheumatoid arthritis, inflammatory boweldiseases (including Crohn's disease), psoriasis, Alzheimer's disease,Parkinson's disease, epilepsy, asthma, sepsis, systemic lupuserythematosus, multiple sclerosis, asthma, rhinitis, cancer andosteoporosis. A compound may be used to treat or prevent rheumatoidarthritis (RA), non specific inflammatory arthritis, erosive bonedisease, chondritis, cartilage degeneration and/or destruction, juvenileinflammatory arthritis, Still's Disease (juvenile and/or adult onset),juvenile idiopathic arthritis, juvenile idiopathic arthritis (botholigoarticular and polyarticular forms), inflammatory bowel diseases(including Crohn's disease, ulcerative colitis, indeterminate colitis,pouchitis), psoriasis, psoriatic arthopathy, ankylosing spondylitis,Sjogren's Disease, Alzheimer's disease (AD), Behcet's Disease,Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), ischemicstroke, pain, epilepsy, osteoporosis, osteopenia, anaemia of chronicdisease, cachexia, diabetes, dyslipidemia, metabolic syndrome, asthma,chronic obstructive airways (or pulmonary) disease, sepsis, fever,respiratory distress syndrome, systemic lupus erythematosus (SLE),multiple sclerosis (MS) immune complex-mediated glomerulonephritis,lupus nephritis (LN), antineutrophil cytoplasmic antibodies (ANCA-)associated glomerulonephritis, minimal change disease, diabeticnephropathy (DN), acute kidney injury (AKI), obstructive uropathy,kidney allograft rejection, cisplatin-induced AKI and obstructiveuropathy, eye diseases (including diabetic retinopathy, diabetic macularoedema, retinopathy of prematurity, age related macular degeneration,macular oedema, proliferative and/or non proliferative retinopathy,corneal vascularisation including neovascularization, retinal veinocclusion, various forms of uveitis and keratitis), thryoiditis,fibrosing disorders including various forms of hepatic fibrosis, variousforms of pulmonary fibrosis, systemic sclerosis, scleroderma, cancer andcancer associated complications (including skeletal complications,cachexia and anaemia).

As discussed above, antibodies of the present invention may be used astarget engagement biomarkers to assess the effectiveness of treatmentwith a compound or complex as described herein. In one embodiment, asample taken from a subject treated with a compound or complex describedherein may be contacted with an antibody of the invention. The antibodymay then be used to determine the amount of TNF superfamilymember-compound complex present within the sample. The amount of complexdetermined using the antibody may be related to the effectiveness of thetreatment. For example, the more complex detected by the antibody of theinvention, the more effective the treatment. The amount of complexdetermined using the antibody is directly proportional to theeffectiveness of the treatment. For example, doubling the amount ofcomplex determined using the antibody may be indicative of a doubling ofthe effectiveness of the treatment.

An antibody of the invention may be used to determine the amount ofcompound-trimer complex using any appropriate technique. Standardtechniques are known in the art and are disclosed herein. For example,ELISA and Western blotting with an antibody of the invention may be usedto determine the amount of compound-trimer complex.

The amount of the compound-trimer complex may be determined by measuringthe mass of the compound-trimer complex, the concentration of thecompound-trimer complex, and the molarity of the compound-trimercomplex. This amount may be given in any appropriate units. For example,the concentration of the compound-trimer complex may be given in pg/ml,ng/ml or μg/ml. The mass of the compound-trimer complex may be given inpg, ng or μg.

The amount of the compound-trimer complex in a sample of interest may becompared with the level of the compound-trimer complex in anothersample, such as a control sample, as described herein. In such a method,the actual amount of the compound-trimer complex, such as the mass,molar amount, concentration or molarity of the compound-trimer complexin the samples may be assessed. The amount of the compound-trimercomplex may be compared with that in another sample without quantifyingthe mass, molar amount, concentration or molarity of the compound-trimercomplex. Thus, the amount of the compound-trimer complex in a sampleaccording to the invention may be assessed as a relative amount, such asa relative mass, relative molar amount, relative concentration orrelative molarity of the compound-trimer complex based on a comparisonbetween two or more samples.

Pharmaceutical Compositions, Dosages and Dosage Regimes

An antibody, compound or complex of the invention may be provided in apharmaceutical composition. The pharmaceutical composition that willnormally be sterile and will typically include a pharmaceuticallyacceptable carrier and/or adjuvant. A pharmaceutical composition of thepresent invention may additionally comprise a pharmaceuticallyacceptable adjuvant and/or carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier may be suitable for parenteral,e.g. intravenous, intramuscular, intradermal, intraocular,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Alternatively, thecarrier may be suitable for non-parenteral administration, such as atopical, epidermal or mucosal route of administration. The carrier maybe suitable for oral administration. Depending on the route ofadministration, the modulator may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

The pharmaceutical compositions of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects.Examples of such salts include acid addition salts and base additionsalts.

Pharmaceutically acceptable carriers comprise aqueous carriers ordiluents. Examples of suitable aqueous carriers that may be employed inthe pharmaceutical compositions of the invention include water, bufferedwater and saline. Examples of other carriers include ethanol, polyols(such as glycerol, propylene glycol, polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. In many cases, it willbe desirable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration.

Pharmaceutical compositions of the invention may comprise additionalactive ingredients.

Also within the scope of the present invention are kits comprisingantibodies, compounds and/or complexes of the invention and instructionsfor use. The kit may further contain one or more additional reagents,such as an additional therapeutic or prophylactic agent as discussedabove.

The compounds identified by the methods and/or antibodies of theinvention and the antibodies of the present invention or formulations orcompositions thereof may be administered for prophylactic and/ortherapeutic treatments.

In therapeutic applications compounds are administered to a subjectalready suffering from a disorder or condition as described above, in anamount sufficient to cure, alleviate or partially arrest the conditionor one or more of its symptoms. Such therapeutic treatment may result ina decrease in severity of disease symptoms, or an increase in frequencyor duration of symptom-free periods. An amount adequate to accomplishthis is defined as a “therapeutically effective amount”.

In prophylactic applications, formulations are administered to a subjectat risk of a disorder or condition as described above, in an amountsufficient to prevent or reduce the subsequent effects of the conditionor one or more of its symptoms. An amount adequate to accomplish this isdefined as a “prophylactically effective amount”. Effective amounts foreach purpose will depend on the severity of the disease or injury aswell as the weight and general state of the subject.

A subject for administration may be a human or non-human animal. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, horses,cows, chickens, amphibians, reptiles, etc. Administration to humans istypical.

A compound or pharmaceutical composition of the invention may beadministered via one or more routes of administration using one or moreof a variety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. Examples of routes of administrationfor compounds or pharmaceutical compositions of the invention includeintravenous, intramuscular, intradermal, intraocular, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection. Alternatively, compound orpharmaceutical composition of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration. The compound or pharmaceutical composition of theinvention may be for oral administration.

A suitable dosage of a compound or pharmaceutical composition of theinvention may be determined by a skilled medical practitioner. Actualdosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A suitable dose may be, for example, in the range of from about 0.01μg/kg to about 1000 mg/kg body weight, typically from about 0.1 μg/kg toabout 100 mg/kg body weight, of the patient to be treated. For example,a suitable dosage may be from about 1 μg/kg to about 10 mg/kg bodyweight per day or from about 10 μg/kg to about 5 mg/kg body weight perday.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single dose may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Administration may be in single or multiple doses. Multiple doses may beadministered via the same or different routes and to the same ordifferent locations. Alternatively, doses can be via a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency may vary depending on the half-life of theantagonist in the patient and the duration of treatment desired.

As mentioned above, compounds or pharmaceutical composition of theinvention may be co-administered with one or other more othertherapeutic agents. For example, the other agent may be an analgesic,anaesthetic, immunosuppressant or anti-inflammatory agent.

Combined administration of two or more agents may be achieved in anumber of different ways. Both may be administered together in a singlecomposition, or they may be administered in separate compositions aspart of a combined therapy. For example, the one may be administeredbefore, after or concurrently with the other.

The following Examples illustrate the invention.

EXAMPLES Example 1—Synthesis of Compounds

Synthesis of compound (1) is disclosed in WO 2013/186229 (Example 44).

Synthesis of compound (2) is disclosed in WO 2013/186229 (Example 89).

Synthesis of compound (3) is disclosed in WO 2014/009295 (Example 129).

Synthesis of compound (4) is disclosed in WO 2014/009295 (Example 173).

Synthesis of compound (5) is disclosed in WO 2014/009295 (Example 319).

Synthesis of compound (6) is disclosed in WO 2013/186229 (Example 490).

Example 2—Antibody Derivation

Following the immunisation of 5 Sprague Dawley rats with human TNFα incomplex with the benzimidazole compound (1), immune B cells werecultured in 96-well plates to induce clonal expansion and antibodysecretion (Tickle, S. et al., High throughput screening for highaffinity antibodies Journal of Laboratory Automation 2009 14: 303-307).Culture supernatants were screened for IgG antibodies preferentiallybinding to human TNFα in complex with compound (1) (at a 50 fold molarexcess), compared to apo human TNFα, in a homogeneous bead-based FMATassay. Human TNFα (+/−compound (1)) was presented on bead surfaces(superavidin-coated Bangs Beads, catalogue number CP01N) by a capturesystem using a human TNF-Receptor I-Fc fusion protein (R&D Systemscatalogue number 372-R1-050), bound with biotinylated anti-human Fc(Jackson catalogue number 109-066-098).

Antibodies which demonstrated preferential binding to the TNFα-compound(1) complex were termed ‘conformation-selective’ and were taken forwardfor cloning. The Fluorescent Foci method (U.S. Pat. No. 7,993,864/EuropeEP1570267B1) was used to identify and isolate antigen-specific B cellsfrom positive wells, and specific antibody variable region genes wererecovered from single cells by reverse transcription (RT)-PCR.

The amino acid sequences of two representative antibodies, CA185_01974and CA185_01979, which demonstrated conformation-selective binding toboth human and mouse TNFα+compound are shown below:

CA185_01974.0 (VR0001837) Light chain variable region (LCVR) (CDRs underlined)  SEQ ID NO: 7DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSL ADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELK Heavy chain variable region (HCVR) (CDRs underlined)  SEQ ID NO: 8DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASI NYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEA YGYNSNWFGYWGQGTLVTVSS  CA185_01979.0 (VR0001842) Light chain variable region (LCVR) (CDRs underlined)  SEQ ID NO: 22DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSL ADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIK Heavy chain variable region (HCVR) (CDRs underlined)  SEQ ID NO: 23EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYIN YSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYH  FDYWGRGVMVTVSS 

Example 3—Potential Epitope of the Derived Antibodies

Given the ability of the rat-derived antibodies to bind to both humanand mouse TNFα in the presence of compounds, detailed analysis of rat,mouse and human amino acid sequences, together with X-ray crystalstructures of TNFα, was undertaken to see if a likely epitope could bedetermined.

Rat UniProt P16599 (SEQ ID NO: 32)        10         20         30         40         50         60 MSTESMIRDV ELAEEALPKK MGGLQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPNK         70         80         90        100        110        120 EEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHVVANHQ AEEQLEWLSQ RANALLANGM        130        140        150        160        170        180 DLKDNQLVVP ADGLYLIYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVS LLSAIKSPCP        190        200        210        220        230 KDTPEGAELK PWYEPMYLGG VFQLEKGDLL SAEVNLPKYL DITESGQVYF GVIAL Mouse UniProt P06804 (SEQ ID NO: 33)        10         20         30         40         50         60 MSTESMIRDV ELAEEALPQK MGGFQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPQR         70         80         90        100        110        120 DEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHVVANHQ VEEQLEWLSQ RANALLANGM        130        140        150        160        170        180 DLKDNQLVVP ADGLYLVYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVN LLSAVKSPCP        190        200        210        220        230 KDTPEGAELK PWYEPIYLGG VFQLEKGDQL SAEVNLPKYL DFAESGQVYF GVIAL Human UniProt P01375  (SEQ ID NO: 34)        10         20         30         40         50         60 MSTESMIRDV ELAEEALPKK TGGPQGSRRC LFLSLFSFLI VAGATTLFCL LHFGVIGPQR         70         80         90        100        110        120 EEFPRDLSLI SPLAQAVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR        130        140        150        160        170        180 DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE        190        200        210        220        230 TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL 

From alignments and comparison of the rat, mouse and human TNFα UniProtsequences, examples of where the rat amino acid sequence differs fromthe human, and where the human and mouse sequences are identical in themature, cleaved product include N168, 1194, F220 and A221 (residues andnumbering from the human sequence).

These residues are highlighted on the crystal structure of human TNFα(1TNF) (FIG. 1). It is possible that any of these amino acids areincluded in the epitope targeted by the antibodies CA185_01974 andCA185_01979.

Following cloning of the antibody variable regions into mouse IgG andmouse Fab (no-hinge) vectors, the conformation-selective nature of thebinding of antibodies CA185_01974 and CA185_01979 was confirmed, using avariety of test compounds bound to TNFα, in HPLC, BIAcore, ELISA andcell-based assays.

Example 4—High Performance Liquid Chromatography (HPLC) to DetermineAntibody Characteristics

Specific binding of mouse Fab fragments was demonstrated by complexformation between CA185_01974 and human TNFα complexed with compound (1)using size exclusion chromatography. Results are shown in FIG. 2. Asshown in this Figure, with a 0.5× molar excess of Fab the predominantpeak corresponds to bound Fab and trimer-compound complex (althoughthere is a small peak showing the presence of some trimer-compoundcomplex not bound to Fab). At a 1.0× molar excess of Fab there is singlehigher molecular weight peak corresponding to Fab bound totrimer-compound complex. At 1.5× and 2× molar excesses of Fab, there isa growing lower molecular peak corresponding to unbound Fab.

The stoichiometry was therefore determined to be 1 Fab: 1 TNFα trimer,with excess Fab appearing at 1.5× and 2× molar excess.

Binding of CA185_01979 to human TNFα complexed with compound (1) wasalso investigated using size exclusion chromatography. Results are shownin FIG. 3. As for CA185_01974, the stoichiometry was determined to be 1Fab: 1 TNFα trimer, with excess Fab appearing at 1.5× and 2× molarexcess.

Example 5—BIAcore Assays to Determine Antibody Characteristics

Surface plasmon resonance was performed at 25° C. using a BIAcore T200(GE Healthcare). Anti-Mouse Fc (Jackson 115-006-071) was immobilised ona CMS Sensor Chip (GE Healthcare) via amine coupling chemistry to acapture level of 6000 response units. HBS-EP buffer (10 mM HEPES pH 7.4,0.15 M NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20—GE Healthcare)+1%DMSO was used as the running buffer. A 10 μl injection of each IgG at 1μg/ml was used for capture by the immobilised anti-mouse Fc to createthe TNFα-binding surface. Human or mouse TNFα (in-house) at 50 nM waspre-incubated with 2 μM compound in HBS-EP+ (1% DMSO) for 5 hours.

A 3 minute injection of human or mouse TNFα+/−test compound was passedover each captured IgG at a flow rate of 30 μl/min. The surface wasregenerated at a flow-rate of 100 min by a 60 s injection of 40 mM HCl×2and a 30 s 5 mM NaOH. Double referenced background subtracted bindingcurves were analysed using the T200 Evaluation software (version 1.0)following standard procedures. Kinetic parameters were determined fromthe fitting algorithm.

The kinetic binding data for human and mouse TNFα in the presence andabsence of test compounds from two chemical series are shown in Tables 1and 2 below.

TABLE 1 BIAcore data with human TNFα ka Antibody Human TNFα (M⁻¹s⁻¹) kd(s⁻¹) KD (M) CA185_01974 + compound (2) 4.2 × 10⁵ 3.9 × 10⁻⁵ 9.4 × 10⁻¹¹CA185_01974 + compound 1 3.2 × 10⁵ 3.8 × 10⁻⁵ 1.2 × 10⁻¹⁰ CA185_01974apo 6.6 × 10⁴ 1.3 × 10⁻³ 1.9 × 10⁻⁸  CA185_01979 + compound (2) 5.7 ×10⁵ 3.3 × 10⁻⁵ 5.8 × 10⁻¹¹ CA185_01979 + compound (1) 4.7 × 10⁵ 1.6 ×10⁻⁵ 3.4 × 10⁻¹¹ CA185_01979 apo 1.1 × 10⁵ 7.1 × 10⁻⁴ 6.7 × 10⁻⁹ 

Both CA185_01974 and CA185_01979 demonstrated >2 log selective bindingfor compound-distorted human TNFα, with representative test compoundsfrom two chemical series.

TABLE 2 BIAcore data with mouse TNFα ka Antibody Mouse TNFα (M⁻¹s⁻¹) kd(s⁻¹) KD (M) CA185_01974 + compound (2) 6.7 × 10⁴ 4.8 × 10⁻⁵ 7.1 × 10⁻¹⁰CA185_01974 + compound (1) 5.8 × 10⁴ 8.8 × 10⁻⁵ 1.5 × 10⁻⁹  CA185_01974apo 4.2 × 10⁴ 4.9 × 10⁻³ 1.2 × 10⁻⁷  CA185_01979 + compound (2) 1.9 ×10⁵ 3.5 × 10⁻⁵ 1.9 × 10⁻¹⁰ CA185_01979 + compound (1) 1.6 × 10⁵ 6.3 ×10⁻⁵ 3.8 × 10⁻¹⁰ CA185_01979 apo 7.2 × 10⁴ 2.0 × 10⁻³ 2.7 × 10⁻⁸ 

Both CA185_01974 and CA185_01979 demonstrated >1.5 and >2 log selectivebinding for compound-distorted mouse TNFα, with representative testcompounds from two chemical series.

Example 6—ELISAs to Determine Antibody Characteristics

A sandwich ELISA was developed to measure the concentration of TNFαbound to compounds of the invention, using antibody CA185_01974.0 thatspecifically detects the conformation of TNFα when in complex with thesecompounds. Briefly, a microtitre plate was coated with CA185_01974.0 toimmobilise TNFα in complex with a test compound. TNFα was incubatedovernight at 28° C. with a 50× molar excess of the test compound.Following this overnight incubation, TNFα was serially diluted in neathuman plasma depleted of endogenous TNFα, in the presence ofheterophilic antibody blockers, and added to the coated plate. Curveswere generated with a concentration range of 0.78 pg/ml-50 pg/ml TNFα. Abiotinylated polyclonal anti TNFα antibody was used to detect boundTNFα, with streptavidin-peroxidase and TMB substrate to give acolorimetric signal. Sensitivity of the assay was increased with the useof tyramide signal amplification, using the ELAST kit from Perkin Elmer,as an additional step between streptavidin-peroxidase and the substrate.

An ELISA was also developed to measure total TNFα (free TNFα+TNFα incomplex with a test compound) in parallel. For this assay the coatingantibody was replaced with a commercial anti-TNFα polyclonal antibody(Invitrogen AHC3812). The sample incubation time was also increased to 3hours. All other steps were identical to the conformation-specificassay. This enables the amount of TNFα in complex with a test compoundto be calculated as a proportion of total TNFα.

Results for the total TNFα ELISA with compounds (3), (4) and (5) areshown in FIG. 4.

Results of the conformation specific TNFα ELISA with CA185_01974.0 andcompounds (3), (4) and (5) are shown in FIG. 5. Apo TNFα gave no signalin this assay, demonstrating the specific nature of the binding ofantibody CA185_01974 to compound-bound TNFα. The antibody was able torecognise TNFα bound by a variety of test compounds from differentchemical series.

Example 7—Cell-Based Assays to Determine Antibody Characteristics

Recombinant antibodies were also tested for binding tocompound-distorted TNFα in a FACS assay using human embryonic kidney(HEK) Jumpin cells, which overexpress TNF-RI after induction withdoxycycline at 1 μg/ml for 2.5 hours. HEK cells were trypsinised andincubated for 2 h in medium to allow recovery of digested TNFRI levels.Human TNFα at 2 μg/mL was pre-incubated with 40 μM compound (1) or 0.4%DMSO for 1 h at 37° C. The preincubation mix was added to the cells for1 h on ice (dilution 1:4, final concentrations: 0.5 μg/mLhuman-TNFα+/−10 μM compound (1) or 0.1% DMSO). Cells were washed, fixed(1.5% PFA) and stained with 1 or 10 μg/mL antibody for 1 h on ice.(Secondary antibody: anti-mouse-Alexa488), before analysis forreceptor-bound TNFα.

As shown in FIG. 6, FACS histogram plots of staining with CA185_01974and CA185_01979 at 1 and 10 μg/ml demonstrate that the antibodies onlyrecognise TNFα which has been pre-incubated with compound (1). There isno staining with the DMSO control.

In addition, specific binding of CA185_01974 and CA185-01979 Fabfragments was demonstrated with compound-distorted membrane-bound TNFα.An engineered NS0 cell line, which overexpresses membrane TNFα, due toknock-out of the TACE cleavage site was incubated with 0.001-10 μMcompound (1) or 0.1% DMSO for 1 h at 37° C. Cells were washed, fixed andstained with antibody Fab fragments at 0.01 or 0.1 μg/ml for 1 hour onice. (Secondary antibody was anti-mouse Fab-DyeLight488 from JacksonImmunoResearch).

FACS histogram plots of staining with CA185_01974 (FIG. 7) andCA185_01979 Fab fragments demonstrate that the antibodies only recogniseTNF which has been pre-incubated with compound (1). There is no stainingwith the DMSO controls.

Example 8—Antibody CA185_01974, Shows a 300-Fold Selectivity for HumanTNF-Compound (4) Complex

Compound (4) was incubated with human and cynomolgus TNF and titratedover mouse full length antibody CA185_01974 to determine an accurateaffinity value. The experiment included the following controls: (i)human or cynomolgus TNF+DMSO over 1974; (ii) human or cynomolgusTNF+DMSO over no antibody; and (iii) human or cynomolgus TNF+compound(4) over no antibody. Each sample and control was carried out induplicate and used four concentrations in each replicate.

As shown in FIGS. 8 and 9, background binding of hTNF+compound (4) andcTNF+compound (4) increased by 5-10RU over the course of the assay. Thisis seen in the higher response of h/cTNF+compound (4) binding toCA185_01974 in the second duplicate. Binding of hTNF and cTNF in theabsence of compound (4) was consistently very low.

Kinetics of hTNF+DMSO binding mouse full length IgG CA185_1974_P8 wasvery similar in this assay to previous single concentration analysis.Affinity of cynomolgus TNF for mouse full length IgG CA185_1974_P8 issimilar, however the kinetics differ.

Table 3 gives the kinetics of each analyte binding to CA185_1974_P8.Table 4 gives the average values and the fold difference+/−compound (4)of TNF kinetics for CA185_1974_p8. FIG. 8 gives the sensograms of bothduplicates of cTNF+/−compound (4). FIG. 9 gives the sensograms of bothduplicates of hTNF+/−compound (4).

TABLE 3 Binding kinetics of hTNF and cTNF +/− compound (4) to theCA185_1974_P8 antibody Duplicate Antibody Analyte ka (1/Ms) kd (1/s) KD(M) KD (pM) 1 CA185_1974_P8 cyno TNF 1.03E+05 1.87E−03 1.83E−08 18270 2CA185_1974_P8 cyno TNF 1.25E+05 1.92E−03 1.54E−08 15350 1 CA185_1974_P8cyno TNF + 1.84E+05 1.46E−05 7.91E−11 79.1 compound (4) 2 CA185_1974_P8cyno TNF + 2.01E+05 2.06E−05 1.03E−10 103 compound (4) 1 CA185_1974_P8human TNF 8.02E+04 1.77E−03 2.21E−08 22100 2 CA185_1974_P8 human TNF1.05E+05 1.67E−03 1.59E−08 15900 1 CA185_1974_P8 human TNF + 3.06E+051.00E−05 3.27E−11 32.7 compound (4) 2 CA185_1974_P8 human TNF + 3.07E+052.73E−05 8.88E−11 88.8 compound (4)

TABLE 4 Average values and fold differences +/− compound (4) of hTNF andcTNF kinetics for CA185_1974_p8. Average of ka kd KD KD duplicates(1/Ms) (1/s) (M) (pM) cyno TNF 1.14E+05 1.90E−03 1.68E−08 16810 cynoTNF + 1.93E+05 1.76E−05 9.09E−11 90.92 compound (4) Fold difference 1.69107.81 184.89 184.89 human TNF 9.27E+04 1.72E−03 1.90E−08 19000 humanTNF + 3.06E+05 1.86E−05 6.08E−11 60.76 compound (4) Fold difference 3.31 92.43 312.70 312.70

Conclusions

The antibodies CA185_01974 and CA185_01989 have been demonstratedspecifically to bind to a compound-distorted state of TNFα, and will beuseful target-engagement biomarkers for compounds of the invention.

The antibodies have been shown to bind to a conformation of TNFα, whichis specifically stabilised by compounds from different chemical series.It is envisaged that these antibodies will become standards in definingthis, and closely related, biologically relevant conformations, of theTNFα trimer, which are stabilised by a wider range of chemical seriesthan are described here. Based on the data shown, the human TNFα trimercould be considered to be stabilised in the defined, biologicallyrelevant conformation described if either CA185_01974 or CA185_01989antibody binds with a K_(D) better than 1 nM in the BIAcore assay formatdescribed above.

Example 9—Compounds and Complexes of Ma et al (2014) and Silvian et al(2011) have Different Characteristics to Those of the Present Invention

As described on page 12458 of Ma et al. (2014) JBC 289:12457-12466, C87was discovered through virtual screening by attempting to find moleculeswhich fit the space occupied by a 7 amino-acid peptide fromloop2/domain2 of TNFR1 in its interaction with the external surface ofTNFβ. The C87 compound from Ma et al. and the BIO8898 compound fromSilvian et al. (2011) ACS Chemical Biology 6:636-647 were tested by thepresent inventors.

Summary of Findings

The Biacore observations described in Ma et al. for C87 could not berepeated.

No evidence of TNF specific inhibition in cells was observed.

Additionally C87 was not observed to bind by mass spectrometry, which issensitive to millimolar affinities.

Extensive crystallography trials only produced apo-TNF (TNF withoutcompound).

In the fluorescence polarisation (FP) assay, C87 showed no significantinhibition above the interference level of the compound with thefluorescent read-out.

Thermo fluor, which measures stabilisation of the thermal meltingtemperature of TNFα, did show a small stabilisation for C87.

In summary, no evidence was found that C87 binds in the centre of thetrimer.

The overwhelming majority of the data suggested no direct interactionwith TNFα. BIO8898 was also found not to bind to TNFα.

Cells—TNF Induced HEK NFKB Reporter Gene Assay

C87 was preincubated with TNFα for 1 hour prior to the addition toHEK-293 cells stably transfected with SEAP under the control of NFκB. Anappropriate counter-screen was also tested in order to detect non-TNFrelated (off target) activity. The assay was incubated overnight beforeinhibition was measured compared to 100% blocking by a control compound.The maximum C87 concentration was 10,000 nM, with a 3-fold serialdilution.

No inhibitory effect could be detected that could not be attributed tooff-target activity.

Biacore

TNF was immobilised using an avi-tag linker and C87 was passed over thechip. In one experiment, a dose response of C87 from a highestconcentration of 10 μM was performed. No binding was observed.

In a second experiment, the flow rate of C87 passing over the chip wasreduced. A small shift was observed but overall binding was negligible.

The binding of C87 to TNF described in Ma et al was likely to besuper-stoichiometric based on the RU value on the Y-axis. At standardTNF density on the chip this value was in the region of thirty timeshigher than expected for simple 1:1 binding.

In another experiment, BIO8898 was tested against the immobilisedsoluble form of CD40L and the soluble form of TNFα by SPR on a Biacore4000 machine. A geomean IC50 of 17 μM was determined for binding againstCD40L whereas no binding was detected at a concentration of up to 100 μMfor TNFα in this assay.

Mass Spectrometry

There was no evidence of C87 binding to human TNFα (20 μM) at aconcentration of 400 μM. A species of lower molecular weight (˜473 Daappears to bind at less than 5% occupancy). C87 has a molecular weightof 503 Da. Based on the occupancy at a concentration of 400 μM, anaffinity of the low molecular weight species in excess of 1 mM ispredicted.

Crystallography

Overall a large effort was put into crystallising C87 with TNFα,including testing conditions that routinely work with compoundsdescribed in the present application. This comprised setting up a largenumber of crystallization trials at different ligand concentrations,different protein concentrations, and different soaking times. A fewcrystals were observed that, on analysis, proved to be salt or TNF withno compound.

Fluorescent Polarization (FP)

C87 was preincubated with TNFα for 1 hour prior to assay against thefluorescent compound (probe). Competition with the fluorescent compoundeither directly (binding at the same site) or indirectly (disruptingTNF) is detected by a reduction in FP.

Extrapolation of the inhibition curve produced an IC50 of about 100 μM.Fluorescence quenching was, however, observed at the highestconcentrations of inhibitor which, when subtracted, resulted innegligible inhibition of C87 in this assay.

Thermofluor

Thermofluor measures the change of melting temperature (Tm) of TNFα dueto compound either stabilising or disrupting the protein. Astabilization effect of 3.8° C. was observed at a concentration of 500μM C87, suggesting the possibility of weak binding, which may not bespecific.

Sequence listing  (LCDR1 of 1974)  SEQ ID NO: 1 QASQDIGN (LCDR2 of 1974)  SEQ ID NO: 2 GATSLAD  (LCDR3 of 1974)  SEQ ID NO: 3LQGQSTPYT  (HCDR1 of 1974)  SEQ ID NO: 4 AYYMA  (HCDR2 of 1974) SEQ ID NO: 5 ASINYDGANTFYRDSVKG  (HCDR3 of 1974)  SEQ ID NO: 6EAYGYNSNWFGY  (LCVR of 1974)  SEQ ID NO: 7DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGT QYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELK  (HCVR of 1974)  SEQ ID NO: 8DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFT VSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSS (LCVR DNA of 1974)  SEQ ID NO: 9GACATCCAGATGACCCAGTCTCCTGCCTCCCTGCCTGCATCCCCGGAAGAAATTGTCACCATCACATGC CAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCGCCTCAGCTC CTGATCTATGGTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGCCAGTAGATCTGGCACA CAGTACTCTCTTAAGATCAGCAGACTGCAGGTTGAAGATTTTGGAATCTTTTACTGTCTACAGGGTCAA AGTACTCCGTACACGTTTGGAGCTGGGACCAAGCTGGAACTGAAA  (HCVR DNA of 1974) SEQ ID NO: 10GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCA GCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAG TGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACT GTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCC ACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACT CTGGTCACTGTCTCGAGC  (1974 LC kappa full)  SEQ ID NO: 11DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGT QYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNN FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNEC  (1974 HC mIgG1 full)  SEQ ID NO: 12DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFT VSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSSAKTTPPSVYPLAPGSAA QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWF VDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSN WEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK  (1974 HC mFabno hinge full) SEQ ID NO: 13DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFT VSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSSAKTTPPSVYPLAPGSAA QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDC  (1974 LC DNA kappa full)  SEQ ID NO: 14GACATCCAGATGACCCAGTCTCCTGCCTCCCTGCCTGCATCCCCGGAAGAAATTGTCACCATCACATGC CAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCGCCTCAGCTC CTGATCTATGGTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGCCAGTAGATCTGGCACA CAGTACTCTCTTAAGATCAGCAGACTGCAGGTTGAAGATTTTGGAATCTTTTACTGTCTACAGGGTCAA AGTACTCCGTACACGTTTGGAGCTGGGACCAAGCTGGAACTGAAACGTACGGATGCTGCACCAACTGTA TCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC TTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGAC GAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAG AGCTTCAACAGGAATGAGTGT  (1974 HC DNA mIgG1 full)  SEQ ID NO: 15GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCA GCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAG TGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACT GTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCC ACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACT CTGGTCACTGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCC CAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACC TGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACT CTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCAC CCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGT ACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTG ACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTT GTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGC TCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAG GTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACA GACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAAC ACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAAC TGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAG AGCCTCTCCCACTCTCCTGGTAAA  (1974 HC DNA mFabno hinge full)  SEQ ID NO: 16GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCA GCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAG TGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACT GTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCC ACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACT CTGGTCACTGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCC CAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACC TGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACT CTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCAC CCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT  (LCDR2 of 1979) SEQ ID NO: 17 GTTSLAD  (LCDR3 of 1979)  SEQ ID NO: 18 LQAYSTPFTF (HCDR1 of 1979)  SEQ ID NO: 19 NSYWD  (HCDR2 of 1979)  SEQ ID NO: 20YINYSGSTGYNPSLKS  (HCDR3 of 1979)  SEQ ID NO: 21 GTYGYNAYHFDY (LCVR of 1979)  SEQ ID NO: 22DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGT QYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIK  (HCVR of 1979)  SEQ ID NO: 23EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISI SRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSS  (LCVR DNA of 1979) SEQ ID NO: 24GACATCCAAATGACACAGTCTCCTGCCTCCCTGTCTGCATCTCTGGAAGAAATTGTCACCATTACATGC CAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCTCCTCACCTC CTGATCTATGGTACCACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGTACA CAGTATTCTCTTAAGATCAGCGGACTACAGGTTGCAGATATTGGAATCTATGTCTGTCTACAGGCTTAT AGTACTCCATTCACGTTCGGCTCAGGGACAAAGCTGGAAATAAAA  (HCVR DNA of 1979) SEQ ID NO: 25GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCT GTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAG TGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATT AGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACA TATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATG GTCACAGTCTCGAGC  (1979 LC Kappa full)  SEQ ID NO: 26DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGT QYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNN FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNEC  (1979 HC mIgG1 full)  SEQ ID NO: 27EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISI SRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSSAKTTPPSVYPLAPGSAAQ TNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHP ASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQV YTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNW EAGNTFTCSVLHEGLHNHHTEKSLSHSPGK  (1979 HC mFabno hinge full) SEQ ID NO: 28EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISI SRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSSAKTTPPSVYPLAPGSAAQ TNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHP ASSTKVDKKIVPRDC  (1979 LC DNA Kappa full)  SEQ ID NO: 29GACATCCAAATGACACAGTCTCCTGCCTCCCTGTCTGCATCTCTGGAAGAAATTGTCACCATTACATGC CAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCTCCTCACCTC CTGATCTATGGTACCACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGTACA CAGTATTCTCTTAAGATCAGCGGACTACAGGTTGCAGATATTGGAATCTATGTCTGTCTACAGGCTTAT AGTACTCCATTCACGTTCGGCTCAGGGACAAAGCTGGAAATAAAACGTACGGATGCTGCACCAACTGTA TCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC TTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGAC GAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAG AGCTTCAACAGGAATGAGTGT  (1979 HC DNA mIgG1 full)  SEQ ID NO: 30GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCT GTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAG TGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATT AGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACA TATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATG GTCACAGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAA ACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGG AACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTG AGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCG GCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACA GTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACT CCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTA GATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCA GTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGT GCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTG TACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGAC TTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACT CAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGG GAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGC CTCTCCCACTCTCCTGGTAAA  (1979 HC DNA mFabno hinge full)  SEQ ID NO: 31GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCT GTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAG TGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATT AGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACA TATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATG GTCACAGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAA ACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGG AACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACTCTG AGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCG GCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT  Rat TNFα  SEQ ID NO: 32MSTESMIRDVELAEEALPKKMGGLQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPNKEEKFPNGLP LISSMAQTLTLRSSSQNSSDKPVAHVVANHQAEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLIY SQVLFKGQGCPDYVLLTHTVSRFAISYQEKVSLLSAIKSPCPKDTPEGAELKPWYEPMYLGGVFQLEKG DLLSAEVNLPKYLDITESGQVYFGVIAL  Mouse TNFα  SEQ ID NO: 33MSTESMIRDVELAEEALPQKMGGFQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPQRDEKFPNGLP LISSMAQTLTLRSSSQNSSDKPVAHVVANHQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLVY SQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELKPWYEPIYLGGVFQLEKG DQLSAEVNLPKYLDFAESGQVYFGVIAL  Human TNFα  SEQ ID NO: 34MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSL ISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQV LFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDR LSAEINRPDYLDFAESGQVYFGIIAL  Soluble form of human TNFα  SEQ ID NO: 35SVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEIN RPDYLDFAESGQVYFGIIAL  Soluble form of human TNFα, but lacking the “S”cloning artefact of SEQ ID NO: 35  SEQ ID NO: 36VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVL FKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRL SAEINRPDYLDFAESGQVYFGIIAL 

1. An antibody that selectively binds to a complex comprising (i) atrimeric protein that is a TNF superfamily member and (ii) a compoundthat is capable of binding to a trimeric protein that is a TNFsuperfamily member, whereby the compound-trimer complex binds to therequisite TNF superfamily receptor and modulates the signalling inducedby the trimer through the receptor.
 2. The antibody of claim 1, whereinthe compound antagonises the signalling induced by the trimer throughthe receptor.
 3. The antibody of claim 2, wherein the compound increasesthe stability of the trimeric form of the TNF superfamily membercompared to the stability of the trimeric form of the TNF superfamilymember in the absence of the compound.
 4. The antibody of claim 3,wherein the increase in stability results in an increase in the thermaltransition midpoint (T_(m)) of the trimeric form of the TNF superfamilymember of at least 1° C.
 5. The antibody of claim 4, wherein theincrease in stability results in an increase in the thermal transitionmidpoint (T_(m)) of the trimeric form of the TNF superfamily member ofat least 10° C.
 6. The antibody of claim 5, wherein the increase in theT_(m) of the trimeric form of the TNF superfamily member is between 10°C. and 20° C.
 7. The antibody of any one of the preceding claims,wherein the compound increases the binding affinity of the TNFsuperfamily member to the requisite receptor compared to the bindingaffinity of the TNF superfamily member to its receptor in the absence ofthe compound.
 8. The antibody of claim 7, wherein the compound increasesthe binding affinity of the TNF superfamily member to the requisitereceptor by increasing the on rate (k_(on-r)) and/or decreasing the offrate (k_(off-r)) compared to the k_(on-r) and k_(off-r) values forbinding of the TNF superfamily member to its receptor in the absence ofthe compound.
 9. The antibody of claim 8, wherein the compound increasesthe binding affinity of the TNF superfamily member to the requisitereceptor by increasing the on rate (k_(on-r)) compared to the k_(on-r)value for binding of the TNF superfamily member to its receptor in theabsence of the compound.
 10. The antibody of any one of claims 7 to 9,wherein the compound decreases the K_(D-r) of the TNF superfamily memberto the requisite receptor compared to the K_(D-r) of the TNF superfamilymember to its receptor in the absence of the compound, wherein: a) thecompound decreases the K_(D-r) of the TNF superfamily member to therequisite receptor by at least 10 times compared to the K_(D-r) of theTNF superfamily member to its receptor in the absence of the compound;b) the K_(D-r) value of the TNF superfamily member for binding to therequisite receptor in the presence of the compound is less than 10 nM.11. The antibody of any one of claims 7 to 9, wherein the compounddecreases the K_(D-r) of the TNF superfamily member to the requisitereceptor compared to the K_(D-r) of the TNF superfamily member to itsreceptor in the absence of the compound, wherein: a) the compounddecreases the K_(D-r) of the TNF superfamily member to the requisitereceptor by at least 4 times compared to the K_(D-r) of the TNFsuperfamily member to its receptor in the absence of the compound; b)the K_(D-r) value of the TNF superfamily member for binding to therequisite receptor in the presence of the compound is less than 600 pM.12. The antibody of claim 11, wherein the K_(D-r) value of the TNFsuperfamily member for binding to the requisite receptor in the presenceof the compound is less than 200 pM.
 13. The antibody of any one of thepreceding claims, wherein said compound has an IC₅₀ value of 500 nM orless.
 14. The antibody of any one of the preceding claims, wherein thecompound is selected from the group consisting of compounds (1)-(6), orsalts or solvates thereof:


15. The antibody of any one of the preceding claims, wherein theantibody binds selectively to the trimer-compound complex compared withbinding to the compound in the absence of the TNF superfamily membertrimer and/or binding to the TNF superfamily member trimer in theabsence of the compound.
 16. The antibody of claim 15, wherein theantibody binds to the trimer-compound complex with a K_(D-ab) that is atleast 100 times lower than the K_(D-ab) for binding to the trimeric TNFsuperfamily member in the absence of compound and/or for binding to thecompound in the absence of the TNF superfamily member.
 17. The antibodyof claim 16, wherein the antibody binds to the trimer-compound complexwith a K_(D-ab) that is at least 200 times lower than the K_(D-ab) forbinding to the trimeric TNF superfamily member in the absence ofcompound and/or for binding to the compound in the absence of the TNFsuperfamily member.
 18. The antibody of any one of the preceding claims,wherein the TNF superfamily member is TNFα and the receptor is the TNFreceptor.
 19. The antibody of claim 18, wherein the receptor is TNFR1.20. An antibody that selectively binds to a complex comprising (i) ahuman TNFα and (ii) a compound selected from the group consisting ofcompounds (1)-(6), or salts or solvates thereof.
 21. The antibody ofclaim 20, wherein the TNFα is TNFα_(s).
 22. The antibody of claim 21,wherein the TNFα_(s) comprises the sequence of SEQ ID NO: 35 or SEQ IDNO: 36, or a variant thereof.
 23. The antibody of any one of claims 20to 22, wherein the TNFα is trimeric.
 24. The antibody of any one ofclaims 20 to 23, wherein the antibody binds selectively to theTNFα-compound complex compared with binding to the compound in theabsence of the TNFα and/or binding to the TNFα in the absence of thecompound.
 25. The antibody of claim 24, wherein the antibody binds tothe TNFα-compound complex with a K_(D-ab) that is at least 100 timeslower than the K_(D-ab) for binding to the TNFα in the absence ofcompound and/or for binding to the compound in the absence of the TNFα.26. The antibody of claim 25, wherein the antibody binds to theTNFα-compound complex with a K_(D-ab) that is at least 200 times lowerthan the K_(D-ab) for binding to the TNFα in the absence of compoundand/or for binding to the compound in the absence of the TNFα.
 27. Theantibody of any one of the preceding claims, which comprises at leastone heavy chain complementarity determining region (HCDR) sequenceselected from SEQ ID NOs: 4-6 and 19-21 and/or at least one light chaincomplementarity determining region (LCDR) sequence selected from SEQ IDNOs: 1-3, 17 and
 18. 28. The antibody of claim 27, which comprises aHCDR3 sequence of SEQ ID NO: 6 or SEQ ID NO:
 21. 29. The antibody ofclaim 27 or 28, which comprises HCDR1, HCDR2 and HCDR3 sequences andLCDR1, LCDR2, and LCDR3 sequences contained within a heavy chainvariable region (HCVR) and light chain variable region (LCVR) pair ofSEQ ID NOs: 8/7 or SEQ ID NOs: 23/22.
 30. The antibody of claim 29,wherein the HCDR1/HCDR2/HCDR3 sequence combination is selected from SEQID NOs: 4/5/6 and SEQ ID NOs: 19/20/21, and/or the LCDR1/LCDR2/LCDR3sequence combination is selected from SEQ ID NOs: 1/2/3 and SEQ ID NOs:1/17/18.
 31. The antibody of any one of claims 27 to 30, which comprisesa HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequence combination of SEQ IDNOs: 4/5/6/1/2/3 or SEQ ID NOs: 19/20/21/1/17/18.
 32. The antibody ofany one of claims 27 to 31, which comprises a heavy chain variableregion (HCVR) sequence of SEQ ID NO: 8 or 23 and/or a light chainvariable region (LCVR) sequence of SEQ ID NO: 7 or 22, or sequenceswhich are at least 95% identical thereto.
 33. The antibody of claim 32,which comprises a HCVR and LCVR sequence pair of SEQ ID NOs: 8/7 or SEQID NOs: 23/22, or sequences which are at least 95% identical thereto.34. The antibody of claim 33, wherein: (a) theHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs:4/5/6/1/2/3 and the remainder of the HCVR and LCVR comprise at least 95%identity to SEQ ID NOs: 8 and 7 respectively; or (b) theHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs:19/20/21/1/17/18 and the the remainder of the HCVR and LCVR comprise atleast 95% identity to SEQ ID NOs: 23 and 22 respectively.
 35. Theantibody of claim 32, which comprises a heavy chain of SEQ ID NO: 12,13, 27 or 28 and/or a light chain of SEQ ID NO: 11 or 26, or sequenceswhich are at least 95% identical thereto.
 36. The antibody of claim 35,which comprises a heavy and light chain pair of SEQ ID NOs: 12/11,13/11, 27/26 or 28/26, or sequences which are at least 95% identicalthereto.
 37. The antibody of claim 36, wherein: (a) theHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs:4/5/6/1/2/3 and the remainder of the heavy and light chains comprise atleast 95% identity to SEQ ID NOs: 12 and 11 respectively; (b) theHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs:4/5/6/1/2/3 and the remainder of the heavy and light chains comprise atleast 95% identity to SEQ ID NOs: 13 and 11 respectively; (c) theHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs:19/20/21/1/17/18 and the remainder of the heavy and light chainscomprise at least 95% identity to SEQ ID NOs: 27 and 26 respectively; or(d) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ IDNOs: 19/20/21/1/17/18 and the remainder of the heavy and light chainscomprise at least 95% identity to SEQ ID NOs: 28 and 26 respectively.38. An antibody which competes for binding to TNFα with, or binds to thesame epitope on TNFα as, an antibody as defined in any one of claims 27to
 37. 39. An antibody according to any one of the preceding claims,which is a humanised antibody.
 40. An antibody according to any one ofthe preceding claims, which is a Fab, modified Fab, Fab′, modified Fab′,F(ab′)₂, Fv, single domain antibody or an scFv.
 41. An isolatedpolynucleotide encoding an antibody as defined in any one of claims 1 to40.
 42. An antibody as defined in any one of claims 1 to 40 for use in amethod of treatment of the human or animal body by therapy.
 43. Apharmaceutical composition comprising an antibody as defined in any oneof claims 1 to 40 and a pharmaceutically acceptable adjuvant and/orcarrier.
 44. Use of an antibody as defined in any one of claims 1 to 40as a target engagement biomarker for the detection of a compound-trimercomplex in a sample obtained from a subject; wherein said antibody isdetectable and said complex comprises a trimeric protein that is a TNFsuperfamily member and a compound that is capable of binding to atrimeric protein that is a TNF superfamily member, whereby thecompound-trimer complex binds to the requisite TNF superfamily receptorand modulates the signalling induced by the trimer through the receptor.45. A method of detecting target engagement of a compound to a trimericTNF superfamily member, whereby the compound-trimer complex binds to therequisite receptor and modulates the signalling induced by the trimerthrough the receptor, said method comprising: (a) obtaining a samplefrom a subject administered said compound; (b) contacting an antibody asdefined in any one of claims 1 to 40 to said sample and a controlsample, wherein said antibody is detectable; (c) determining the amountof binding of said detectable antibody to said sample and said controlsample, wherein binding of said detectable antibody to said samplegreater than binding of said detectable antibody to said control sampleindicates target engagement of said compound to said trimeric TNFsuperfamily member.
 46. Use of an antibody as defined in any one ofclaims 1 to 40 in screening for a compound that elicits a conformationalchange in a trimeric TNF superfamily member, wherein said conformationalchange modulates the signalling of the requisite TNF superfamilyreceptor on binding of the trimeric TNF superfamily member.
 47. Acomplex comprising a trimeric protein that is a TNF superfamily memberand a compound that is bound thereto, whereby the compound-trimercomplex binds to the requisite TNF superfamily receptor and modulatesthe signalling induced by the trimer through the receptor, wherein saidcomplex binds to an antibody as defined in any one of claims 1 to 40with a K_(D-ab) of 1 nM or less.
 48. The complex of claim 47, whereinthe TNF superfamily member is TNFα.
 49. A TNFα trimer, said TNFα trimerbeing able to bind TNFR1, but wherein signalling from said bound TNFR1is attenuated or antagonised, wherein said TNFα trimer binds to eitheror both of the following antibodies with a a K_(D-ab) of 1 nM or less:(i) an antibody with a heavy chain of SEQ ID NO: 27 and a light chain ofSEQ ID NO: 26; or (ii) an antibody with a heavy chain of SEQ ID NO: 12and a light chain of SEQ ID NO:
 11. 50. A TNFα trimer according to claim49, wherein the TNFα subunits comprise the amino acid sequence of SEQ IDNO: 36, or a corresponding sequence.
 51. A compound that is capable ofbinding to a trimeric protein that is a TNF superfamily member to form acomplex, whereby the compound-trimer complex binds to the requisite TNFsuperfamily receptor and modulates the signalling induced by the trimerthrough the receptor, wherein the compound-trimer complex binds to anantibody as defined in any one of claims 1 to 40 with a K_(D-ab) of 1 nMor less.
 52. The compound of claim 51, wherein the TNF superfamilymember is TNFα.
 53. A complex according to claim 47 or 48, a trimeraccording to claim 49 or 50, or a compound according to claim 51 or 52for use in a method of therapy practised on the human or animal body.54. The complex, trimer or compound for use according to claim 53, foruse in the treatment and/or prevention of one or more of autoimmune andinflammatory disorders; neurological and neurodegenerative disorders;pain and nociceptive disorders; and cardiovascular disorders.
 55. Thecomplex, trimer or compound for use according to claim 54, for use inthe treatment and/or prevention of one or more of rheumatoid arthritis,Crohn's disease, psoriasis, systemic lupus erythematosus, Alzheimer'sdisease, Parkinson's disease and epilepsy.
 56. A method of treatingand/or preventing one of more of autoimmune and inflammatory disorders;neurological and neurodegenerative disorders; pain and nociceptivedisorders; and cardiovascular disorders, by directly or indirectlyadministering to a patient in need thereof a complex according to claim47 or 48, a trimer according to claim 49 or 50, or a compound accordingto claim 51 or
 52. 57. The method of claim 56, wherein one or more ofrheumatoid arthritis, Crohn's disease, psoriasis, systemic lupuserythematosus, Alzheimer's disease, Parkinson's disease and epilepsy aretreated and/or prevented.
 58. The complex, trimer or compound for useaccording to any one of claims 53-55, or the method of claim 56 or 57,wherein the therapy is on the human body or the patient is a human. 59.A method of identifying a compound that is capable of binding to atrimeric protein that is a TNF superfamily member and modulatingsignalling of the trimeric protein through the receptor, comprising thesteps of: (a) performing a binding assay to measure the binding affinityof a test compound-trimer complex comprising a trimeric protein that isa TNF superfamily member and a test compound to an antibody thatselectively binds to said complex; (b) comparing the binding affinity asmeasured in step (a) with the binding affinity of a differentcompound-trimer complex known to bind with high affinity to the antibodyreferred to in step (a); and (c) selecting the compound present in thecompound-trimer complex of step (a) if its measured binding affinity isacceptable when considered in the light of the comparison referred to instep (b).
 60. The method of claim 59, wherein the antibody bindsselectively to the TNF trimer-compound complex compared with binding tothe compound in the absence of the TNF superfamily member trimer and/orbinding to the TNF superfamily member trimer in the absence of thecompound.
 61. The method of claim 60, wherein the antibody binds to theTNF superfamily member trimer-compound complex with a K_(D-ab) that isat least 100 times lower than the K_(D-ab) for binding to the TNFsuperfamily member trimer in the absence of compound and/or for bindingto the compound in the absence of the TNF superfamily member trimer. 62.The method of claim 61, wherein the antibody binds to the TNFsuperfamily member trimer-compound complex with a K_(D-ab) that is atleast 200 times lower than the K_(D-ab) for binding to the TNFsuperfamily member trimer in the absence of compound and/or for bindingto the compound in the absence of the TNF superfamily member trimer. 63.The method of any one of claims 59 to 62, wherein the antibody is asdefined in any one of claims 1 to
 40. 64. The method of any one ofclaims 59 to 63, which is a high throughput assay.
 65. The method of anyone of claims 59 to 64, wherein the test compound increases thestability of the trimeric form of the TNF superfamily member compared tothe stability of the trimeric form of the TNF superfamily member in theabsence of the compound.
 66. The method of claim 65, wherein theincrease in stability results in an increase in the thermal transitionmidpoint (T_(m)) of the trimeric form of the TNF superfamily member ofat least 1° C.
 67. The method of claim 66, wherein the increase instability results in an increase in the thermal transition midpoint(T_(m)) of the trimeric form of the TNF superfamily member of at least10° C.
 68. The method of claim 67, wherein the increase in the T_(m) ofthe trimeric form of the TNF superfamily member is between 10° C. and20° C.
 69. The method of any one claims 59 to 68, wherein the testcompound increases the binding affinity of the TNF superfamily member tothe requisite receptor compared to the binding affinity of the TNFsuperfamily member to its receptor in the absence of the compound. 70.The method of claim 69, wherein the test compound increases the bindingaffinity of the TNF superfamily member to the requisite receptor byincreasing the on rate (k_(on-r)) and/or decreasing the off rate(k_(off-r)) compared to the k_(on-r) and k_(off-r) values for binding ofthe TNF superfamily member to its receptor in the absence of thecompound.
 71. The method of claim 69, wherein the test compoundincreases the binding affinity of the TNF superfamily member to therequisite receptor by increasing the on rate (k_(on-r)) compared to thek_(on-r) value for binding of the TNF superfamily member to its receptorin the absence of the compound.
 72. The method of claim 69, 70 or 71,wherein the test compound decreases the K_(D-r) of the TNF superfamilymember to the requisite receptor compared to the K_(D-r) of the TNFsuperfamily member to its receptor in the absence of the compound,wherein: a) the compound decreases the K_(D-r) of the TNF superfamilymember to the requisite receptor by at least 10 times compared to theK_(D-r) of the TNF superfamily member to its receptor in the absence ofthe compound; b) the K_(D-r) value of the TNF superfamily member forbinding to the requisite receptor in the presence of the compound isless than 10 nM.
 73. The method of claim 69, 70 or 71, wherein the testcompound decreases the K_(D-r) of the TNF superfamily member to therequisite receptor compared to the K_(D-r) of the TNF superfamily memberto its receptor in the absence of the compound, wherein: a) the compounddecreases the K_(D-r) of the TNF superfamily member to the requisitereceptor by at least 4 times compared to the K_(D-r) of the TNFsuperfamily member to its receptor in the absence of the compound; b)the K_(D-r) value of the TNF superfamily member for binding to therequisite receptor in the presence of the compound is less than 600 pM.74. The method of claim 73, wherein the K_(D-r) value of the TNFsuperfamily member for binding to the requisite receptor in the presenceof the compound is less than 200 pM.
 75. The method of any one claims 59to 74, wherein said test compound has an IC₅₀ value of 500 nM or less.76. The method of any one of claims 59 to 75, wherein the compound instep (b) is selected from the group consisting of compounds (1)-(6), orsalts or solvates thereof.
 77. The method of any one of claims 59 to 76,wherein the TNF superfamily member is TNFα and the receptor is the TNFreceptor.